ITI-D1 Kunitz domain mutants as hNE inhibitors

ABSTRACT

Mutants of Kunitz domain 1 (ITI-D1) of human inter-α-trypsin inhibitor (ITI), are useful as inhibitors of human neutrophil elastase. Mutants characterized by one or more of the following substitutions (numbered to correspond to bovine pancreatic trypsin inhibitor, the archetypal Kunitz domain) are of particular interest: (a) Val15 or Ile15, (b) Ala16, (c) Phe18, (d) Pro19, (e) Arg1, (f) Pro2, and/or (g) Phe4.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of Ser. No. 08/849,406 filedJul. 21, 1999, now pending, which is a national stage of PCT/US95/16349filed Dec. 15, 1995, which is a continuation-in-part of application Ser.No. 08/358,160 filed Dec. 16, 1994, now patented (U.S. Pat. No.5,663,143), which is a continuation-in-part of application Ser. No.08/133,031 filed Feb. 28, 1992, now abandoned, which is the nationalstage of PCT/US92/01501, filed Feb. 28, 1992.

[0002] While PCT/US92/01501 was filed as a continuation-in-part ofLadner, Guterman, Roberts, Markland, Ley, and Kent, Ser. No. 07/664,989,now patented (U.S. Pat. No. 5,223,409), which is a continuation-in-partof Ladner, Guterman, Roberts, and Markland, Ser. No. 07/487,063, filedMar. 2, 1990, now abandoned, which is a continuation-in-part of Ladnerand Guterman, Ser. No. 07/240,160, filed Sept. 2, 1988, now abandoned,the instant application does not claim §120 benefit prior toPCT/US92/01501.

[0003] All of the foregoing applications, whether or not §120 benefit isclaimed, are hereby incorporated by reference.

[0004] The following related and commonly-owned applications are alsoincorporated by reference:

[0005] Robert Charles Ladner, Sonia Kosow Guterman, Rachel BaribaultKent, and Arthur Charles Ley are named as joint inventors on U.S. Ser.No. 07/293,980, filed Jan. 8, 1989, and entitled GENERATION ANDSELECTION OF NOVEL DNA-BINDING PROTEINS AND POLYPEPTIDES. Thisapplication has been assigned to Protein Engineering Corporation.

[0006] Robert Charles Ladner, Sonia Kosow Guterman, and Bruce LindsayRoberts are named as a joint inventors on a U.S. Ser. No. 07/470,651filed Jan. 26, 1990 (now abandoned), entitled “PRODUCTION OF NOVELSEQUENCE-SPECIFIC DNA-ALTERING ENZYMES”, likewise assigned to ProteinEngineering Corp.

[0007] Ladner, Guterman, Kent, Ley, and Markland, Ser. No. 07/558,011 isalso assigned to Protein Engineering Corporation.

[0008] Ladner filed an application on May 17, 1991, Ser. No. 07/715,934that is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0009] Field of the Invention

[0010] This invention relates to novel proteins that inhibit humanneutrophil elastase (hNE). A large fraction of the sequence of each ofthese proteins is identical to a known human protein which has verylittle or no inhibitory activity with respect to hNE.

[0011] Information Disclosure Statement

[0012] 1. hNE , Its Natural Inhibitors, and Pathologies

[0013] Human Neutrophil Elastase (hNE, also known as Human LeukocyteElastase (hLE); EC 3.4.21.11) is a 29 Kd protease with a wide spectrumof activity against extracellular matrix components (CAMP82, CAMP88,MCWH89). The enzyme is one of the major neutral proteases of theazurophil granules of polymorphonuclear leucocytes and is involved inthe elimination of pathogens and in connective tissue restructuring(TRAV88). In cases of hereditary reduction of the circulatingα-1-protease inhibitor (API, formerly known as al antitrypsin), theprincipal systemic physiological inhibitor of hNE (HEID86), or theinactivation of API by oxidation (“smoker's emphysema”), extensivedestruction of lung tissue may result from uncontrolled elastolyticactivity of hNE (CANT89). Several human respiratory disorders, includingcystic fibrosis and emphysema, are characterized by an increasedneutrophil burden on the epithelial surface of the lungs (SNID91,MCEL91, GOLD86) and hNE release by neutrophils is implicated in theprogress of these disorders (MCEL91, WEIS89). A preliminary study ofaerosol administration of API to cystic fibrosis patients indicates thatsuch treatment can be effective both in prevention of respiratory tissuedamage and in augmentation of host antimicrobial defenses (MCEL91).

[0014] API presents some practical problems to large-scale routine useas a pulmonary anti-elastolytic agent. These include the relativelylarge size of the molecule (394 residues, 51 k Dalton), the lack ofintramolecular stabilizing disulfide bridges, and specific posttranslational modifications of the protein by glycosylation at threesites. Perhaps of even greater importance is the sensitivity of API tooxidation, such as those released by activated neutrophils. Hence asmall stable nontoxic highly efficacious inhibitor of hNE would be ofgreat therapeutic value.

[0015] 2. Proteinaceous Serine Protease Inhibitors.

[0016] A large number of proteins act as serine protease inhibitors byserving as a highly specific, limited proteolysis substrate for theirtarget enzymes. In many cases, the reactive site peptide bond (“scissilebond”) is encompassed in at least one disulfide loop, which insures thatduring conversion of virgin to modified inhibitor the two peptide chainscannot dissociate.

[0017] A special nomenclature has evolved for describing the active siteof the inhibitor. Starting at the residue on the amino side of thescissile bond, and moving away from the bond, residues are named P1, P2,P3, etc. (SCHE67). Residues that follow the scissile bond are calledP1′, P2′, P3′, etc. It has been found that the main chain of proteininhibitors having very different overall structure are highly similar inthe region between P3 and P3′ with especially high similarity for P2, P₁and P1′ (LASK80 and works cited therein). It is generally accepted thateach serine protease has sites S1, S2, etc. that receive the side groupsof residues P1, P2, etc. of the substrate or inhibitor and sites S1′,S2′, etc. that receive the side groups of P1′, P2′, etc. of thesubstrate or inhibitor (SCHE67). It is the interactions between the Ssites and the P side groups that give the protease specificity withrespect to substrates and the inhibitors specificity with respect toproteases.

[0018] The serine protease inhibitors have been grouped into familiesaccording to both sequence similarity and the topological relationshipof their active site and disulfide loops. The families include thebovine pancreatic trypsin inhibitor (Kunitz), pancreatic secretorytrypsin inhibitor (Kazal), the Bowman-Birk inhibitor, and soybeantrypsin inhibitor (Kunitz) families. Some inhibitors have severalreactive sites on a single polypeptide chains, and these distinctdomains may have different sequences, specificities, and eventopologies.

[0019] One of the more unusual characteristics of these inhibitors istheir ability to retain some form of inhibitory activity even afterreplacement of the P1 residue. It has further been found thatsubstituting amino acids in the P₅ to P₅% region, and more particularlythe P3 to P3′ region, can greatly influence the specificity of aninhibitor. LASK80 suggested that among the BPTI (Kunitz) family,inhibitors with P1 Lys and Arg tend to inhibit trypsin, those withP1=Tyr, Phe, Trp, Leu and Met tend to inhibit chymotrypsin, and thosewith P1=Ala or Ser are likely to inhibit elastase. Among the Kazalinhibitors, they continue, inhibitors with P1=Leu or Met are stronginhibitors of elastase, and in the Bowman-Kirk family elastase isinhibited with P1 Ala, but not with P1 Leu.

[0020] “Kunitz” Domain Proteinase Inhibitors.

[0021] Bovine pancreatic trypsin inhibitor (BPTI, a.k.a. aprotonin) is a58 a.a. serine proteinase inhibitor of the BPTI (Kunitz) domain (KuDom)family. Under the tradename TRASYLOL, it is used for countering theeffects of trypsin released during pancreatitis. Not only is its 58amino acid sequence known, the 3D structure of BPTI has been determinedat high resolution by X-ray diffraction (HUBE77, MARQ83, WLOD84,WLOD87a, WLOD87b), neutron diffraction (WLOD84), and by NMR (WAGN87).One of the X-ray structures is deposited in the Brookhaven Protein DataBank as “6PTI” [sic]. The 3D structure of various BPTI homologues(EIGE90, HYNE90) are also known. At least sixty homologues have beenreported; the sequences of 39 homologues are given in Table 13, and theamino acid types appearing at each position are compiled in Table 15.The known human homologues include domains of Lipoprotein AssociatedCoagulation Inhibitor (LACI) (WUNT88, GIRA89), Inter-α-Trypsin Inhibitor(ALBR83a, ALBR83b, DIAR90, ENGH89, TRIB86, GEBH86, GEBH90, KAUM86,ODOM90, SALI90), and the Alzheimer beta-Amyloid Precursor Protein.Circularized BPTI and circularly permuted BPTI have binding propertiessimilar to BPTI (GOLD83). Some proteins homologous to BPTI have more orfewer residues at either terminus.

[0022] In BPTI, the P1 residue is at position 15. Tschesche et al.(TSCH87) reported on the binding of several BPTI P1 derivatives tovarious proteases: Dissociation constants for BPTI P1 derivatives,Molar. Residue Trypsin Chymotrypsin Elastase Elastase #15 (bovine(bovine (porcine (human P1 pancreas) pancreas) pancreas) leukocytes)lysine 6.0 · 10⁻¹⁴ 9.0 · 10⁻⁹ − 3.5 · 10⁻⁶ (WT) glycine − − + 7.0 · 10⁻⁹alanine + − 2.8 · 10⁻⁸ 2.5 · 10⁻⁹ valine − − 5.7 · 10⁻⁸ 1.1 · 10⁻¹⁰leucine − − 1.9 · 10⁻⁸ 2.9 · 10⁻⁹

[0023] From the report of Tschesche et al. we infer that molecular pairsmarked “+” have K_(d)s≧3.5·10⁻⁶ M and that molecular pairs marked “−”have K_(d)s>>3.5·10⁻⁶ M. It is apparent that wild-type BPTI has onlymodest affinity for hNE, however, mutants of BPTI with higher affinityare known. While not shown in the Table, BPTI does not significantlybind hCG. However, Brinkmann and Tschesche (BRIN90) made a triple mutantof BPTI (viz. K15F, R17F, M52E) that has a Ki with respect to hCG of5.0×10-7 M.

[0024] b 3. ITI Domain 1 and ITI Domain 2 as an Initial Protein BindingDomains (IPBD)

[0025] Many mammalian species have a protein in their plasma that can beidentified, by sequence homology and similarity of physical and chemicalproperties, as inter-α-trypsin inhibitor (ITI), a large (M_(r) ca240,000) circulating protease inhibitor (for recent reviews see ODOM90,SALI90, GEBH90, GEBH86). The sequence of human ITI is shown in Table400. The intact inhibitor is a glycoprotein and is currently believed toconsist of three glycosylated subunits that interact through a strongglycosaminoglycan linkage (ODOM90, SALI90, ENGH89, SELL87). Theanti-trypsin activity of ITI is located on the smallest subunit (ITIlight chain, unglycosylated M_(r) ca 15,000) which is identical in aminoacid sequence to an acid stable inhibitor found in urine (UTI) and serum(STI) (GEBH86, GEBH90). The amino-acid sequence of the ITI light chainis shown in Table 400. The mature light chain consists of a 21 residueN-terminal sequence, glycosylated at Ser₁₀, followed by two tandemKunitz-type domains the first of which is glycosylated at Asn₄₅(ODOM90). In the human protein, the second Kunitz-type domain has beenshown to inhibit trypsin, chymotrypsin, and plasmin (ALBR83a, ALBR83b,SELL87, SWAI88). The first domain lacks these activities but has beenreported to inhibit leukocyte elastase (≈1 μM>K_(i)≈1 nM) (ALBR83a,b,ODOM90). cDNA encoding the ITI light chain also codes forα-1-microglobulin (TRAB86, KAUM86, DIAR90); the proteins are separatedpost-translationally by proteolysis.

[0026] The two Kunitz domains of the ITI light chain (ITI-D1 and ITI-D2)possesses a number of characteristics that make them useful as InitialPotential Binding Domains (IPBDs). ITI-DL comprises at least residues 26to 76 of the UTI sequence shown in FIG. 1 of GEBH86. The Kunitz domaincould be thought of as comprising residues from as early as residue 22to as far as residue 79. Residues 22 through 79 constitute a58-amino-acid domain having the same length as bovine pancreatic trypsininhibitor (BPTI) and having the cysteines aligned. ITI-D2 comprises atleast residues 82 through 132; residues as early as 78 and as later as135 could be included to give domains closer to the classical58-amino-acid length. As the space between the last cysteine of ITI-D1(residue 76 of ITI light chain) and the first cysteine of ITI-D2(residue 82 of ITI light chain) is only 5 residues, one can not assign58 amino acids to each domain without some overlap. Unless otherwisestated, herein, we have taken the second domain to begin at residue 78of the ITI light chain. Each of the domains are highly homologous toboth BPTI and the EpiNE series of proteins described in U.S. Pat. No.5,223,409. Although x-ray structures of the isolated domains ITI-D1 andITI-D2 are not available, crystallographic studies of the relatedKunitz-type domain isolated from the Alzheimer's amyloid β-protein(AAβP) precursor show that this polypeptide assumes a 3D structurealmost identical to that of BPTI (HYNE90).

[0027] The three-dimensional structure of α-dendrotoxin from green mambavenom has been determined (SKAR92) and the structure is highly similarto that of BPTI. The author states, “Although the main-chain fold ofα-DTX is similar to that of homologous bovine pancreatic trypsininhibitor (BPTI), there are significant differences involving segmentsof the polypeptide chain close to the ‘antiprotease site’ of BPTI.Comparison of the structure of α-DTX with the existing models of BPTIand its complexes with trypsin and kallikrein reveals structuraldifferences that explain the inability of α-DTX to inhibit trypsin andchymotrypsin.”

[0028] The structure of the black mamba K venom has been determined byNMR spectroscopy and has a 3D structure that is highly similar to thatof BPTI despite 32 amino-acid sequence differences between residues 5and 55 (the first and last cysteines)(BERN93). “The solution structureof Toxin K is very similar to the solution structure of the basicpancreatic trypsin inhibitor (BPTI) and the X-ray crystal structure ofthe α-dendrotoxin from Dendroaspis angusticeps (α-DTX), with r.m.s.d.values of 1.31 Å and 0.92 Å, respectively, for the backbone atoms ofresidues 2 to 56. Some local structural differences between Toxin K andBPTI are directly related to the fact that intermolecular interactionswith two of the four internal molecules of hydration water in BPTI arereplaced by intramolecular hydrogen bonds in Toxin K.” Thus, it islikely that the solution 3D structure of either of the isolated ITI-D1domain or of the isolated ITI-D2 domain will be highly similar to thestructures of BPTI, AAβP, and black mamba K venom. In this case, theadvantages described previously for use of BPTI as an IPBD apply toITI-D1 and to ITI-D2. ITI-D1 and ITI-D2 provide additional advantages asan IPBD for the development of specific anti-elastase inhibitoryactivity. First, the ITI-D1 domain has been reported to inhibit bothleukocyte elastase (ALBR83a,b, ODOM90) and Cathepsin-G (SWAI88, ODOM90);activities which BPTI lacks. Second, ITI-D1 lacks affinity for therelated serine proteases trypsin, chymotrypsin, and plasmin (ALBR83a,b,SWAI88), an advantage for the development of specificity in inhibition.ITI-D2 has the advantage of not being glycosylated. Additionally, ITI-Dland ITI-D2 are human-derived polypeptides so that derivatives areanticipated to show minimal antigenicity in clinical applications.

[0029] 4. Secretion of Heteroloqous Proteins from Pichia pastoris

[0030] Others have produced a number of proteins in the yeast Pichiapastoris. For example, Vedvick et al. (VEDV91) and Wagner et al.(WAGN92) produced aprotinin from the alcohol oxidase promoter withinduction by methanol as a secreted protein in the culture medium (CM)at ≈1 mg/mL. Gregg et al. (GREG93) have reviewed production of a numberof proteins in P. pastoris. Table 1 of GREG93 shows proteins that havebeen produced in P. pastoris and the yields.

[0031] 5. Recombinant Production of Kunitz Domains:

[0032] Aprotinin has been made via recombinant-DNA technology (AUER87,AUER88, AUER89, AUER90, BRIN90, BRIN91, ALTM91).

[0033] 6. Construction Methods:

[0034] Unless otherwise stated, genetic constructions and othermanipulations are carries out by standard methods, such as found instandard references (e.g. AUSU87 and SAMB89).

[0035] No admission is made that any cited reference is prior art orpertinent prior art, and the dates given are those appearing on thereference and may not be identical to the actual publication date. Thedescriptions of the teachings of any cited reference are based on ourpresent reading thereof, and we reserve the right to revise thedescription if an error comes to our attention, and to challenge whetherthe description accurately reflects the actual work reported. We reservethe right to challenge the interpretation of cited works, particularlyin light of new or contradictory evidence.

SUMMARY OF THE INVENTION

[0036] The present invention describes a series of small potentproteinaceous inhibitors of human neutrophil elastase (hNE). One groupof inhibitors is derived from a Kunitz-type inhibitory domain found in aprotein of human origin, namely, the light chain of humanInter-α-trypsin inhibitor (ITI) which contains domains designated ITI-D1and ITI-D2. The present invention discloses variants of ITI-D1 andITI-D2 that have very high affinity for hNE. The present inventioncomprises modifications to the ITI-D2 sequence that facilitate itsproduction in the yeast Pichia pastoris and that are highly potentinhibitors of hNE. The invention also relates to methods of transferringsegments of sequence from one Kunitz domain to another and to methods ofproduction.

[0037] The invention is presented as a series of examples that describedesign, production, and testing of actual inhibitors and additionalexamples describing how other inhibitors could be discovered. Theinvention relates to proteins that inhibit human neutrophil elastase(hNE) with high affinity. NOMENCLATURE and ABBREVIATIONS Term Meaningx::y Fusion of gene x to gene y in frame. X::Y Fusion protein expressedfrom x::y fusion gene. μM Micromolar, 10⁻⁶ molar. nM Namomolar, 10⁻⁹molar. pM Picomolar, 10⁻¹² molar. Single-letter amino-acid codes: A: AlaC: Cys D: Asp E: Glu F: Phe G: Gly H: His I: Ile K: Lys L: Leu M: Met N:Asn P: Pro Q: Gln R: Arg S: Ser T: Thr V: Val W: Trp Y: Tyr

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0038] A protein sequence can be called an “aprotinin-like Kunitzdomain” if it contains a sequence that when aligned to minimizemismatches, can be aligned, with four or fewer mismatches, to thepattern:Cys-(Xaa)₆-Gly-Xaa-Cys-(Xaa)₈-[Tyr|Phe]-(Xaa)₆-Cys-(Xaa)₂-Phe-Xaa-[Tyr|Trp|Phe]-Xaa-Gly-Cys-(Xaa)₄-[Asn|Gly]-Xaa-[Phe|Tyr]-(Xaa)₅-Cys-(Xaa)₃-Cys(SEQ ID NO:86), where bracketed amino acids separated by a | symbol arealternative amino acids for a single position. For example, [Tyr|Phe]indicates that at that position, the amino acid may be either Tyr orPhe. The symbol Xaa denotes that at that position, any amino acid may beused. For the above test, an insertion or deletion counts as onemismatch.

[0039] In aprotonin, the cysteines are numbered 5, 14, 30, 38, 51, and55 and are joined by disulfides 5-to-55, 14-to-38, and 30-to-51. Residue15 is called the P1 residue (SCHE67); residues toward the amino terminusare called P2 (residue 14), P3 (residue 13), etc. Residue 16 is calledP1′, 17 is P2′, 18 is P3′, etc.

[0040] There are many homologues of aprotonin, which differ from it atone or more positions but retain the fundamental structure definedabove. For a given list of homologues, it is possible to tabulate thefrequency of occurrence of each amino acid at each ambiguous position.(The sequence having the most prevalent amino acid at each ambiguousposition is listed as “Consensus Kunitz Domain” in Table 100).

[0041] A “human aprotonin-like Kunitz domain” is an aprotonin-likeKunitz domain which is found in nature in a human protein. Humanaprotonin-like Kunitz domains include, but are not limited to, ITI-D1,ITI-D2, App-I, TFPI2-D1, TFPI2-D2, TFPI2-D3, LACI-D1, LACI-D2, LACI-D3,A3 collagen, and the HKI B9 domain. In this list, D1, D2, etc., denotethe first, second, etc. domain of the indicated multidomain protein.

[0042] “Weak”, “Moderate”, “Strong” and “Very Strong” binding to andinhibition of hNE are defined in accordance with Table 55. Preferably,the proteins of the present invention have a Ki of less than 1000 pM(i.e., are “strong” inhibitors), more preferably less than 50 pM, mostpreferably less than 10 pM (i.e., are “very strong” inhibitors).

[0043] For purposes of the present invention, an aprotonin-like Kunitzdomain may be divided into ten segments, based on the consensus sequenceand the location of the catalytic site. Using the amino acid numberingscheme of aprotonin, these segments are as follows (see Table 100):

[0044] 1: 1-4 (residues before first Cys)

[0045] 2: 5-9 (first Cys and subsequent residues before P6)

[0046] 3: 10-13 (P6 to P3)

[0047] 4: 14 (second Cys; P2)

[0048] 5: 15-21 (P1, and P1′ to P6′)

[0049] 6: 22-30 (after P6 and up to and incl. third Cys.)

[0050] 7: 31-36 (after third Cys and up to consensus Gly-Cys)

[0051] 8: 37-38 (consensus Gly-Cys)

[0052] 9: 39-42 (residues after Gly-Cys and before consensus [Asn|Gly]

[0053] 10: 43-55 (up to last Cys)(also includes residues after last Cys,if any)

[0054] It will be appreciated that in those aprotonin-like Kunitzdomains that differ from aprotonin by one or more amino acid insertionsor deletions, or which have a different number of amino acids before thefirst cysteine or after the last cysteine, the actual amino acidposition may differ from that given above. It is applicant's intent thatthese domains be numbered so as to correspond to the aligned aprotoninsequence, e.g., the first cysteine of the domain is numbered amino acid5, for the purpose of segment identification. Note that segment 1, whilea part of aprotonin, is not a part of the formal definition of anaprotonin-like Kunitz domain, and therefore it is not required that theproteins of the present invention include a sequence corresponding tosegment 1. Similarly, part of segment 10 (after the last Cys) is not arequired part of the domain.

[0055] A “humanized inhibitor” is one in which at least one of segments3, 5, 7 and 9 differs by at least one nonconservative modification fromthe most similar (based on amino acid identities) human aprotonin-likeKunitz domain, at least one of segments 2, 6, and 10 (considered up tothe last Cys) is identical, or differs only by conservativemodifications, from said most similar human aprotonin-like Kunitzdomain, and which is not-identical to any naturally occurring nonhumanaprotonin-like Kunitz domain. (Note that segment 1 is ignored in makingthis determination since it is outside the sequence used to define adomain, and segments 4 and 8 are ignored because they are required bythe definition of an aprotonin-like Kunitz domain.)

[0056] The proteins of the present invention are preferably humanizedstrong or very strong hNE inhibitors. It should be noted that the humanaprotonin-like Kunitz domains thus far identified are merely weak hNEinhibitors.

[0057] For the purpose of the appended claims, an aprotonin-like Kunitzdomain is “substantially homologous” to a reference domain if, over thecritical region (aprotonin residues 5-55) set forth above, it is atleast at least 50% identical in amino acid sequence to the correspondingsequence of or within the reference domain, and all divergences take theform of conservative and/or semi-conservative modifications.

[0058] Proteins of the present invention include those comprising aKunitz domain that is substantially homologous to the reference proteinsEPI-HNE-3, EPI-HNE-4, DPI.1.1, DPI.1.2, DPI.1.3, DPI.2.1, DPI.2.2,DPI.2.3, DPI.3.1, DPI.3.2, DPI.3.3, DPI.4.1, DPI.4.2, DPI.4.3, DPI.5.1,DPI.5.2, DPI.5.3, DPI.6.1, DPI.6.2, DPI.6.3, DPI.6.4, DPI.6.5, DPI.6.6,DPI.6.7, DPI.7.1, DPI.7.2, DPI.7.3, DPI.7.4, DPI.7.5, DPI.8.1, DPI.8.2,DPI.8.3, DPI.9.1, DPI.9.2, or DPI.9.3, as defined in Table 100.Homologues of EPI-HNE-3 and EPI-HNE-4 are especially preferred.

[0059] Preferably, the hNE-binding domains of the proteins of thepresent invention are at least 80% identical, more preferably, at least90% identical, in amino acid sequence to the corresponding referencesequence. Most preferably, the number of mismatches is zero, one, two,three, four or five. Desirably, the hNE-binding domains diverge from thereference domain solely by one or more conservative modifications.

[0060] “Conservative modifications” are defined as:

[0061] a) conservative substitutions of amino acids as hereafterdefined, and

[0062] b) single or multiple insertions or deletions of amino acids atthe termini, at interdomain boundaries, in loops or in other segments ofrelatively high mobility (as indicated, for example, by high temperaturefactors or lack of resolution in X-ray diffraction, neutron diffraction,or NMR). Preferably, except at the termini, no more than about fiveamino acids are inserted or deleted at a particular locus, and themodifications are outside regions known to contain binding sitesimportant to activity.

[0063] “Conservative substitutions” are herein defined as exchangeswithin on of the following five groups:

[0064] I. Small aliphatic, nonpolar or slightly polar residues: [Ala,Ser, Thr, (Pro, Gly)],

[0065] II. Acidic amino acids and their amides: [Asp, Glu, Asn, Gln],

[0066] III. Polar, positively charged residues: [His, Lys, Arg],

[0067] IV. Aliphatic nonpolar residues: [Met, Leu, Ile, Val, (Cys)], and

[0068] V. Large, aromatic residues: [Phe, Tyr, Trp]

[0069] Residues Pro, Gly, and Cys are parenthesized because they havespecial conformational roles. Cys often participates in disulfide bonds;when not so doing, it is highly hydrophobic. Gly imparts flexibility tothe chain; it is often described as a “helix breaker” although many ahelices contain Gly. Pro imparts rigidity to the chain and is alsodescribed as a “helix breaker”. Although Pro is most often found inturns, Pro is also found in helices and sheets. These residues may beessential at certain positions and substitutable elsewhere.

[0070] Semi-Conservative Modifications” are defined herein astranspositions of adjacent amino acids (or their conservativereplacements), and semi-conservative substitutions. “Semi-conservativesubstitutions” are defined to be exchanges between two of groups (I)-(V)above which are limited either to the supergroup consisting of (I),(II), and (III) or to the supergroup consisting of (IV) and (V). For thepurpose of this definition, however, glycine and alanine are consideredto be members of both supergroups.

[0071] “Non-conservative modifications” are modifications which areneither conservative nor semi-conservative.

[0072] Preferred proteins of the present invention are furthercharacterized by one of more of the preferred, highly preferred, or mostpreferred mutations set forth in Table 711.

[0073] Preferably, the proteins of the present invention havehNE-inhibitory domains which are not only substantially homologous to areference domain, but also qualify as humanized inhibitors.

[0074] Claim 1 of PCT/US92/01501 refers to proteins denoted EpiNEalpha,EpiNE1, EpiNE2, EpiNE3, EpiNE4, EpiNE5, EpiNE6, EpiNE7, and EpiNE8.Claim 3 refers to proteins denoted ITI-E7, BITI-E7, BITI-E&-1222,AMINO1, AMINO2, MUTP1, BITI-E7-141, MUTT26A, MUTQE, and MUT1619. (Withthe exception of EpiNEalpha, the sequences of all of these domainsappears in Table 100.) Claims 4-6 related to inhibitors which arehomologous to, but not identical with, the aforementioned inhibitors.These homologous inhibitors could differ from the lead inhibitors by oneor more class A substitutions (claim 4), one or more class A or Bsubstitutions (claim 5), or one or more class A, B or C substitutions(claim 6). Class A, B and C substitutions were defined in Table 65 ofPCT/US92/01501. For convenience, Table 65 has been duplicated in thisspecification.

[0075] The meaning of classes A, B and C were as follows: A, no majoreffect expected if molecular charge stays in range −1 to +1; B, majoreffects not expected, but more likely than with A; and C, residue inbinding interface, any change must be tested. Each residue position wasassigned an A, B, C or X rating; X meant no substitution allowed. At thenon-X positions, allowed substitutions were noted.

[0076] In one series of embodiments, the present invention is directedto HNE inhibitors as disclosed in Ser. No. 08/133,031 (previouslyincorporated by reference), which is the U.S. national stage ofPCT/US92/01501.

[0077] The invention disclosed in 08/133,031 relates to muteins of BPTI,ITI-D1 and other Kunitz domain-type inhibitors which have a highaffinity for elastase. Some of the described inhibitors are derived fromBPTI and some from ITI-D1. However, hybrids of the identified muteinsand other Kunitz domain-type inhibitors could be constructed.

[0078] For the purpose of simultaneously assessing the affinity of alarge number of different BPTI and ITI-D1 muteins, DNA sequencesencoding the BPTI or ITI-D1 was incorporated into the genome of thebacteriophage M13. The KuDom is displayed on the surface of M13 as anamino-terminal fusion with the gene III coat protein. Alterations in theKuDom amino acid sequence were introduced. Each pure population of phagedisplaying a particular KuDom was characterized with regard to itsinteractions with immobilized hNE or hCG. Based on comparison to the pHelution profiles of phage displaying other KuDoms of known affinitiesfor the particular protease, mutant KuDoms having high affinity for thetarget proteases were identified. Subsequently, the sequences of thesemutant KuDoms were determined (typically by sequencing the correspondingDNA sequence).

[0079] Certain aprotonin-like protease inhibitors were shown to have ahigh affinity for HNE (≈10¹²/M). These 58 amino acid polypeptides werebiologically selected from a library of aprotinin mutants producedthrough synthetic diversity. Positions P1, P1′, P2′, P3′, and P4′ werevaried. At P1, only VAL and ILE were selected, although LEU, PHE, andMET were allowed by the synthetic conditions. At P1′, ALA and GLY wereallowed and both were found in proteins having high affinity. (While notexplored in the library, many Kazal family inhibitors of serineproteases have glutamic or aspartic acid at P1′.) All selected proteinscontained either PHE or MET at P2′; LEU, ILE, and VAL, which are aminoacids with branched aliphatic side groups, were in the library butapparently hinder binding to HNE. Surprisingly, position P3′ of allproteins selected for high affinity for HNE have phenylalanine. No onehad suggested that P3′ was a crucial position for determiningspecificity relative to HNE. At P4′, SER, PRO, THR, LYS, and GLN wereallowed; all of these except THR were observed. PRO and SER are found inthe derivatives having the highest affinity.

[0080] In Ser. No. 08/133,031, Table 61 showed the variability of 39naturally-occurring Kunitz domains. All these proteins have 51 residuesin the region C₅ through C₅₅; the total number of residues varies due tothe proteins having more or fewer residues at the termini. Table 62 listthe names of the proteins that are included in Table 61. Table 64 citesworks where these sequences are recorded. Table 63 shows a histogram ofhow many loci show a particular variability vs. the variability. “Core”refers to residues from 5 to 55 that show greater sequence andstructural similarity than do residues outside the core.

[0081] At ten positions a single amino-acid type is observed in all 42cases, these are C₅, G₁₂, C₁₄, C₃₀, F₃₃, G₃₇, C₃₈, N₄₃, C₅₁, and C₅₅.Although there are reports that each of these positions may besubstituted without complete loss of structure, only G₁₂, C₁₄, G₃₇, andC₃₈ are close enough to the binding interface to offer any incentive tomake changes. G₁₂ is in a conformation that only glycine can attain;this residue is best left as is. Marks et al. (MARK87) replaced both C₁₄and C₃₈ with either two alanines or two threonines. The C₁₄/C₃₈ cystinebridge that Marks et al. removed is the one very close to the scissilebond in BPTI; surprisingly, both mutant molecules functioned as trypsininhibitors. Both BPTI(C14A,C38A) and BPTI(C14T,C38T) are stable andinhibit trypsin. Altering these residues might give rise to a usefulinhibitor that retains a useful stability, and the phage-display of avariegated population is the best way to obtain and test mutants thatembody alterations at either 14 or 38. Only if the C₁₄/C₃₈ disulfide isremoved, would the strict conservation of G37 be removed.

[0082] At seven positions (viz. 23, 35, 36, 40, 41, 45, and 47) only twoamino-acid types have been found. At position 23 only Y and F areobserved; the para position of the phenyl ring is solvent accessible andfar from the binding site. Changes here are likely to exert subtleinfluences on binding and are not a high priority for variegation.Similarly, 35 has only the aromatic residues Y and W; phenylalaninewould probably function well here. At 36, glycine predominates whileserine is also seen. Other amino acids, especially {N, D, A, R}, shouldbe allowed and would likely affect binding properties. Position 40 hasonly G or A; structural models suggest that other amino acids would betolerated, particularly those in the set {S, D, N, E, K, R, L, M, Q, andT}. Position 40 is close enough to the binding site that alteration heremight affect binding. At 41, only N, and K have been seen, but any aminoacid, other than proline, should be allowed. The side group is exposed,so hydrophilic side groups are preferred, especially {D, S, T, E, R, Q,and A}. This residue is far enough from the binding site that changeshere are not expected to have big effects on binding. At 45, F is highlypreferred, but Y is observed once. As one edge of the phenyl ring isexposed, substitution of other aromatics (W or H) is likely to makemolecules of similar structure, though it is difficult to predict howthe stability will be affected. Aliphatics such as leucine or methionine(not having branched C_(β)s) might also work here. At 47, only S and Thave been seen, but other amino acids, especially {N, D, G, and A},should give stable proteins.

[0083] At one position (44), only three amino-acid types have beenobserved. Here, asparagine predominates and may form internal hydrogenbonds. Other amino acids should be allowed, excepting perhaps proline.

[0084] At the remaining 40 positions, four or more amino acids have beenobserved; at 28 positions, eight or more amino-acid types are seen.Position 25 exhibits 13 different types and 5 positions (1, 6, 17, 26,and 34) exhibit 12 types. Proline (the most rigid amino acid) has beenobserved at fourteen positions: 1, 2, 8, 9, 11, 13, 19, 25, 32, 34, 39,49, 57, and 58. The φ,ψ angles of BPTI (CREI84, Table 6-3, p. 222)indicate that proline should be allowed at positions 1, 2, 3, 7, 8, 9,11, 13, 16, 19, 23, 25, 26, 32, 35, 36, 40, 42, 43, 48, 49, 50, 52, 53,54, 56, and 58. Proline occurs at four positions (34, 39, 57, and 58)where the BPTI φ,ψ angles indicate that it should be unacceptable. Weconclude that the main chain rearranges locally in these cases.

[0085] Based on these data and excluding the six cysteines, we judgethat the KuDom structure will allow those substitutions shown in Table65. The class indicates whether the substitutions: A) are very likely togive a stable protein having substantially the same binding to hNE, hCG,or some other serine protease as the parental sequence, B) are likely togive similar binding as the parent, or C) are likely to give a proteinsretaining the KuDom structure, but which are likely to affect thebinding. Mutants in class C must be tested for affinity, which isrelatively easy using a display-phage system, such as the one set forthin W0/02809. The affinity of hNE and hCG inhibitors is most sensitive tosubstitutions at positions 15, 16, 17, 18, 34, 39, 19, 13, 11, 20, 36 ofBPTI, if the inhibitor is a mutant of ITI-D1, these positions must beconverted to their ITI-Dl equivalents by aligning the cysteines in BPTIand ITI-D1.

[0086] Wild-type BPTI is not a good inhibitor of hNE. BPTI with a singleK15L mutation exhibits a moderate affinity for HNE (K_(d)=2.9·10⁻⁹ M)(BECK88b). However, the amino terminal Kunitz domain (BI-8e) of thelight chain of bovine inter-α-trypsin inhibitor has been generated byproteolysis and shown to be a potent inhibitor of HNE (K_(d)=4.4·10⁻¹¹M) (ALBR83).

[0087] It has been proposed that the P1 residue is the primarydeterminant of the specificity and potency of BPTI-like molecules(SINH91, BECK88b, LASK80 and works cited therein). Although both BI-8eand BPTI(K15L) feature LEU at their respective P1 positions, there is a66 fold difference in the affinities of these molecules for HNE. Wetherefore hypothesized that other structural features must contribute tothe affinity of BPTI-like molecules for HNE.

[0088] A comparison of the structures of BI-8e and BPTI(K15L) revealsthe presence of three positively charged residues at positions 39, 41,and 42 of BPTI which are absent in BI-8e. These hydrophilic and highlycharged residues of BPTI are displayed on a loop which underlies theloop containing the P1 residue and is connected to it via a disulfidebridge. Residues within the underlying loop (in particular residue 39)participate in the interaction of BPTI with the surface of trypsin(BLOW72) and may contribute significantly to the tenacious binding ofBPTI to trypsin. These hydrophilic residues might, however, hamper thedocking of BPTI variants with HNE. Supporting this hypothesis, BI-8edisplays a high affinity for HNE and contains no charged residues inresidues 39-42. Hence, residues 39 through 42 of wild type BPTI werereplaced with the corresponding residues (MGNG) of the human homologueof BI-8e. As we anticipated, a BPTI(K15L) derivative containing the MGNG39-42 substitution exhibited a higher affinity for HNE than did thesingle substitution mutant BPTI(Kl5L). Mutants of BPTI with Met atposition 39 are known, but positions 40-42 were not mutatedsimultaneously.

[0089] Tables 207 and 208 present the sequences of additional novel BPTImutants with high affinity for hNE. We believe these mutants to have anaffinity for hNE which is about an order of magnitude higher than thatof BPTI (K15V, R17L). All of these mutants contain, besides the activesite mutations shown in the Tables, the MGNG mutation at positions39-42.

[0090] Although BPTI has been used in humans with very few adverseeffects, a KuDom having much higher similarity to a human KuDom posesmuch less risk of causing an immune response. Thus, we transferred theactive site changes found in EpiNE7 into the first KuDom ofinter-α-trypsin inhibitor. For the purpose of this application, thenumbering of the nucleic acid sequence for the ITI light chain gene isthat of TRAB86 and that of the amino acid sequence is the one shown forUTI in FIG. 1 of GEBH86. The necessary coding sequence for ITI-DI is the168 bases between positions 750 and 917 in the cDNA sequence presentedin TRAB86. The amino acid sequence of human ITI-D1 is 56 amino acidslong, extending from Lys-22 to Arg-77 of the complete ITI light chainsequence. The P1 site of ITI-DI is Met-36. Tables 220-221 presentcertain ITI mutants; note that the residues are numbered according tothe homologus Kunitz domain of BPTI, i.e., with the P1 residue numbered15. It should be noted that it is probably acceptable to truncate theamino-terminal of ITI-D1, at least up to the first residue homologouswith BPTI.

[0091] The EpiNE7-inspired mutation (BPTI 15-19 region) of ITI-D1significantly enhanced its affinity for hNE. We also discovered thatmutation of a different part of the molecule (BPTI 1-4 region) provideda similar increase in affinity. When these two mutational patterns werecombined, a synergistic increase in affinity was observed. Furthermutations in nearby amino acids (BPTI 26, 31, 34) led to additionalimprovements in affinity.

[0092] The elastase-binding muteins of ITI-DI envisioned hereinpreferably differ from the wild-type domain at one or more of thefollowing positions (numbered per BPTI): 1, 2, 4, 15, 16, 18, 19, 31 and34. More preferably, they exhibit one or more of the followingmutations: Lys1→Arg; Glu2→Pro; Ser4→Phe*; Met15→Val*, Ile; Gly16→Ala;THr18→Phe*; Ser19→Pro; Thr26→ALa; Glu31→Gln; Gln34→Val*. Introduction ofone or more of the starred mutations is especially desirable, and, inone preferred embodiment, at least all of the starred mutations arepresent.

[0093] In a second series of embodiments, the present invention relatesto Kunitz-type domains which inhibit HNE, but excludes those domainscorresponding exactly to the lead domains of claims 1 and 3 ofPCT/US92/01501. Preferably, such domains also differ from these leaddomains by one or more mutations which are not class A substitutions,more preferably, not class A or B substitutions, and still morepreferably, not class A, B or C substitutions, as defined in Table 65.Desirably, such domains are each more similar to one of theaforementioned reference proteins than to any of the lead proteins setforth in PCT/US92/01501.

[0094] The examples contain numerous examples of amino-acid sequencesaccompanied by DNA sequences that encode them. It is to be understoodthat the invention is not limited to the particular DNA sequence shown.

EXAMPLE 1 Expression and Display of BPTI, ITI-D1, and Other KunitzDomains.

[0095] Table 30 shows a display gene that encodes: 1) the M13 III signalpeptide, 2) BPTI, and 3) the first few amino-acids of mature M13 IIIprotein. Phage have been made in which this gene is the only iii-likegene so that all copies of III expressed are expected to be modified atthe amino terminus of the mature protein. Substitutions in the BPTIdomain can be made in the cassettes delimited by the AccIII, XhoI,PflMI, ApaI, BssHII, StuI, XcaI, EspI, SphI, or NarI (same recognitionas KasI) sites. Table 100 gives amino-acid sequences of a number ofKunitz domains, some of which inhibit hNE. Each of the hNE-inhibitingsequences shown in Table 100 can be expressed as an intact hNE-bindingprotein or can be incorporated into a larger protein as a domain.Proteins that comprise a substantial part of one of the hNE-inhibitingsequences found in Table 100 are expected to exhibit hNE-inhibitoryactivity. This is particularly true if the sequence beginning with thefirst cysteine and continuing through the last cysteine is retained.

[0096] ITI domain 1 is a Kunitz domain as discussed below. The abilityof display phage to be retained on matrices that display hNE is relatedto the affinity of the particular Kunitz domain (or other protein)displayed on the phage. Expression of the ITI domain 1::iii fusion geneand display of the fusion protein on the surface of phage weredemonstrated by Western analysis and phage titer neutralizationexperiments. The infectivity of ITI-D1-display phage was blocked by upto 99% by antibodies that bind ITI while wild-type phage wereunaffected.

[0097] Table 35 gives the sequence of a fusion gene comprising: a) thesignal sequence of M13 III, b) ITI-D1, and c) the initial part of matureIII of M13. The displayed ITI-D1 domain can be altered by standardmethods including: i) oligonucleotide-directed mutagenesis ofsingle-stranded phage DNA, and ii) cassette mutagenesis of RF DNA usingthe restriction sites (BglI, EagI, NcoI, Styl, PstI, and KasI (twosites)) designed into the gene.

EXAMPLE 2 Fractionation of MA-ITI-D1 Phase Bound to Agarose-ImmobilizedProtease Beads.

[0098] To test if phage displaying the ITI-D1::III fusion proteininteract strongly with the proteases human neutrophil elastase (hNE),aliquots of display phage were incubated with agarose-immobilized hNEbeads (“hNE beads”). The beads were washed and bound phage eluted by pHfractionation as described in US 5,223,409. The pHs used in the stepgradient were 7.0, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, and 2.0.Following elution and neutralization, the various input, wash, and pHelution fractions were titered. Phage displaying ITI-D1 were compared tophage that display EpiNE-7.

[0099] The results of several fractionations are shown in Table 212(EpiNE-7 or MA-ITI-D1 phage bound to hNE beads). The pH elution profilesobtained using the control display phage (EpiNE-7) were similar previousprofiles (US 5,223,409). About 0.3% of the EpiNE-7 display phage appliedto the hNE beads eluted during the fractionation procedure and theelution profile had a maximum for elution at about pH 4.0.

[0100] The MA-ITI-D1 phage show no evidence of great affinity for hNEbeads. The pH elution profiles for MA-ITI-D1 phage bound to hNE beadsshow essentially monotonic decreases in phage recovered with decreasingpH. Further, the total fractions of the phage applied to the beads thatwere recovered during the fractionation procedures were quite low:0.002%.

[0101] Published values of K_(i) for inhibition neutrophil elastase bythe intact, large (M_(r)=240,000) ITI protein range between 60 and 150nM (SWAI88, ODOM90). Our own measurements of pH fraction of displayphage bound to hNE beads show that phage displaying proteins with lowaffinity (>1 μM) for hNE are not bound by the beads while phagedisplaying proteins with greater affinity (nM) bind to the beads and areeluted at about pH 5. If the first Kunitz-type domain of the ITI lightchain is entirely responsible for the inhibitory activity of ITI againsthNE, and if this domain is correctly displayed on the MA-ITI-D1 phage,then it appears that the minimum affinity of an inhibitor for hNE thatallows binding and fractionation of display phage on hNE beads isbetween 50 and 100 nM.

EXAMPLE 3 Alteration of the P1 Region of ITI-D1.

[0102] We assume that ITI-D1 and EpiNE-7 have the same 3D configurationin solution as BPTI. Although EpiNE-7 and ITI-D1 are identical atpositions 13, 17, 20, 32, and 39, they differ greatly in theiraffinities for hNE. To improve the affinity of ITI-D1 for hNE, theEpiNE-7 sequence Val₁₅-Ala₁₆-Met₁₇-Phe₁₈-Pro₁₉-Arg₂₀ (bold, underscoredamino acids are alterations) was incorporated into the ITI-D1 sequenceby cassette mutagenesis between the EagI and Styl/NcoI sites shown inTable 35. Phage isolates containing the ITI-D1::III fusion gene with theEpiNE-7 changes around the P1 position are called MA-ITI-D1E7.

EXAMPLE 4 Fractionation of MA-ITI-D1E7 Phage.

[0103] To test if ITI-D1E7-display phage bind hNE beads, pH elutionprofiles were measured. Aliquots of EpiNE-7, MA-ITI-D1, and MA-ITI-D1E7display phage were incubated with hNE beads for three hours at roomtemperature (RT). The beads were washed and phage were eluted asdescribed in U.S. Pat. No. 5,223,409, except that only three pH elutionswere performed. These data are in Table 215. The pH elution profile ofEpiNE-7 display phage is as described. MA-ITI-D1E7 phage show a broadelution maximum around pH 5. The total fraction of MA-ITI-D1E7 phageobtained on pH elution from hNE beads was about 40-fold less than thatobtained using EpiNE-7 display phage.

[0104] The pH elution behavior of MA-ITI-D1E7 phage bound to hNE beadsis qualitatively similar to that seen using BPTI[K15L]-III-MA phage.BPTI with the K15L mutation has an affinity for hNE of ≈3 nM.(Alterations and mutations are indicated by giving the original(wild-type) amino-acid type, then the position, and then the newamino-acid type; thus K15L means change Lys₁₅ to Leu.) Assuming all elseremains the same, the pH elution profile for MA-ITI-D1E7 suggests thatthe affinity of the free ITI-D1E7 domain for hNE might be in the nMrange. If this is the case, the substitution of the EpiNE-7 sequence inplace of the ITI-D1 sequence around the P1 region has produced a 20- to50-fold increase in affinity for hNE (assuming K_(i)=60 to 150 nM forthe unaltered ITI-D1).

[0105] If EpiNE-7 and ITI-D1E7 have the same solution structure, theseproteins present the identical amino acid sequences to hNE over theinteraction surface. Despite this similarity, EpiNE-7 exhibits a roughly1000-fold greater affinity for hNE than does ITI-D1E7. This observationhighlights the importance of non-contacting secondary residues inmodulating interaction strengths.

[0106] Native ITI light chain is glycosylated at two positions, Ser₁ andAsn₄₅ (GEBH86). Removal of the glycosaminoglycan chains has been shownto decrease the affinity of the inhibitor for hNE about 5-fold (SELL87).Another potentially important difference between EpiNE-7 and ITI-D1E7 isthat of net charge. The changes in BPTI that produce EpiNE-7 reduce thetotal charge on the molecule from +6 to +1. Sequence differences betweenEpiNE-7 and ITI-D1E7 further reduce the charge on the latter to −1.Furthermore, the change in net charge between these two molecules arisesfrom sequence differences occurring in the central portions of themolecules. Position 26 is Lys in EpiNE-7 and is Thr in ITI-D1E7, whileat position 31 these residues are Gln and Glu, respectively. Thesechanges in sequence not only alter the net charge on the molecules butalso position a negatively charged residue close to the interactionsurface in ITI-D1E7. It may be that the occurrence of a negative chargeat position 31 (which is not found in any other of the hNE inhibitorsdescribed here) destabilized the inhibitor-protease interaction.

EXAMPLE 5 Preparation of BITI-E7 Phage

[0107] Possible reasons for MA-ITI-D1E7 phage having lower affinity forhNE than do MA-EpiNE7 phage include: a) incorrect cleavage of theIIIsignal::ITI-D1E7::matureIII fusion protein, b) inappropriate negativecharge on the ITI-D1E7 domain, c) conformational or dynamic changes inthe Kunitz backbone caused by substitutions such as Phe₄ to Ser₄, and d)non-optimal amino acids in the ITI-D1E7:hNE interface, such as Q3₄ orA₁₁.

[0108] To investigate the first three possibilities, we substituted thefirst four amino acids of EpiNE7 for the first four amino acids ofITI-D1E7. This substitution should provide a peptide that can be cleavedby signal peptidase-I in the same manner as is theIIIsignal::EpiNE7::matureIII fusion. Furthermore, Phe₄ of BPTI is partof the hydrophobic core of the protein; replacement with serine mayalter the stability or dynamic character of ITI-DlE7 unfavorably.ITI-D1E7 has a negatively charged Glu at position 2 while EpiNE7 hasPro. We introduced the three changes at the amino terminus of theITI-D1E7 protein (K1R, E2P, and S4F) by oligonucleotide-directedmutagenesis to produce BITI-E7; phage that display BITI-E7 are calledMA-BITI-E7.

[0109] We compared the properties of the ITI-III fusion proteinsdisplayed by phage MA-ITI-D1 and MA-BITI using Western analysis asdescribed previously and found no significant differences in apparentsize or relative abundance of the fusion proteins produced by eitherdisplay phage strain. Thus, there are no large differences in theprocessed forms of either fusion protein displayed on the phage. Byextension, there are also no large differences in the processed forms ofthe gene III fusion proteins displayed by MA-ITI-D1E7 and MA-EpiNE7.Large changes in protein conformation due to altered processing aretherefore not likely to be responsible for the great differences inbinding to hNE-beads shown by MA-ITI-D1E7 and MA-EpiNE7 display phage.

[0110] We characterized the binding properties to hNE-beads of MA-BITIand MA-BITI-E7 display phage using the extended pH fractionationprocedure described in U.S. Pat. No. 5,223,409. The results are in Table216. The pH elution profiles for MA-BITI and MA-BITI-E7 show significantdifferences from the profiles exhibited by MA-ITI-D1 and MA-ITI-D1E7. Inboth cases, the alterations at the putative amino terminus of thedisplayed fusion protein produce a several-fold increase in the fractionof the input display phage eluted from the hNE-beads.

[0111] The binding capacity of hNE-beads for display phage varies amongpreparations of beads and with age for each individual preparation ofbeads. Thus, it is difficult to directly compare absolute yields ofphage from elutions performed at different times. For example, thefraction of MA-EpiNE7 display phage recovered from hNE-beads variestwo-fold among the experiments shown in Tables 212, 215, and 216.However, the shapes of the pH elution profiles are similar. It ispossible to correct somewhat for variations in binding capacity ofhNE-beads by normalizing display phage yields to the total yield ofMA-EpiNE7 phage recovered from the beads in a concurrent elution. Whenthe data shown in Tables 212, 215, and 216 are so normalized, therecoveries of display phage, relative to recovered MA-EpiNE7, are shownin Table 10. TABLE 10 Recovery of Display phage Normalized Display Phagestrain fraction of input MA-ITI-D1 0.0067 MA-BITI 0.018 MA-ITI-D1E70.027 MA-BITI-E7 0.13

[0112] Thus, the changes in the amino terminal sequence of the displayedprotein produce a three- to five-fold increase in the fraction ofdisplay phage eluted from hNE-beads.

[0113] In addition to increased binding, the changes introduced intoMA-BITI-E7 produce phage that elute from hNE-beads at a lower pH than dothe parental MA-ITI-D1E7 phage. While the parental display phage elutewith a broad pH maximum centered around pH 5.0, the pH elution profilefor MA-BITI-E7 display phage has a pH maximum at around pH 4.75 to pH4.5.

[0114] The pH elution maximum of the MA-BITI-E7 display phage is betweenthe maxima exhibited by the BPTI(K15L) and BPTI(K15V, R17L) displayphage (pH 4.75 and pH 4.5 to pH 4.0, respectively) described in U.S.Pat. No. 5,223,409. From the pH maximum exhibited by the display phagewe predict that the BITI-E7 protein free in solution may have anaffinity for hNE in the 100 pM range. This would represent anapproximately ten-fold increase in affinity for hNE over that estimatedabove for ITI-D1E7.

[0115] As was described above, Western analysis of phage proteins showthat there are no large changes in gene III fusion proteins uponalteration of the amino terminal sequence. Thus, it is unlikely that thechanges in affinity of display phage for hNE-beads can be attributed tolarge-scale alterations in protein folding resulting from altered(“correct”) processing of the fusion protein in the amino terminalmutants. The improvements in binding may in part be due to: 1) thedecrease in the net negative charge (−1 to 0) on the protein arisingfrom the Glu to Pro change at position 2, or 2) increased proteinstability resulting from the Ser to Phe substitution at residue 4 in thehydrophobic core of the protein, or 3) the combined effects of bothsubstitutions.

EXAMPLE 6 Production and Properties of MA-BITI-E7-1222 andMA-BITI-E7-141

[0116] Within the presumed Kunitz:hNE interface, BITI-E7 and EpiNE7differ at only two positions: 11 and 34. In EpiNE7 these residues areThr and Val, respectively. In BITI-E7 they are Ala and Gln. In additionBITI-E7 has Glu at 31 while EpiNE7 has Gln. This negative charge mayinfluence binding although the residue is not directly in the interface.We used oligonucleotide-directed mutagenesis to investigate the effectsof substitutions at positions 11, 31 and 34 on the protease:inhibitorinteraction.

[0117] ITI-D1 derivative BITI-E7-1222 is BITI-E7 with the alterationA11T. ITI-D1 derivative BITI-E7-141 is BITI-E7 with the alterations E31Qand Q34V; phage that dhe presence of tisplay these proteins areMA-BITI-E7-1222 and MA-BITI-E7-141. We determined the binding propertiesto hNE-beads of MA-BITI-E7-1222 and MA-BITI-E7-141 display phage usingthe extended pH fractionation protocol described previously. The resultsare in Tables 217 (for MA-BITI-E7 and MA-BITI-E7-1222) and 218 (forMA-EpiNE7 and MA-BITI-E7-141). The pH elution profiles for theMA-BITI-E7 and MA-BITI-E7-1222 phage are almost identical. Both phagestrains exhibit pH elution profiles with identical maxima (between pH5.0 and pH 4.5) as well as the same total fraction of input phage elutedfrom the hNE-beads (0.03%). Thus, the T11A substitution in the displayedITI-D1 derivative has no appreciable effect on the binding to hNE-beads.

[0118] In contrast, the changes at positions 31 and 34 strongly affectthe hNE-binding properties of the display phage. The elution profile pHmaximum of MA-BITI-E7-141 phage is shifted to lower pH relative to theparental MA-BITI-E7 phage. Further, the position of the maximum (betweenpH 4.5 and pH 4.0) is identical to that exhibited by MA-EpiNE7 phage inthis experiment. Finally, the MA-BITI-E7-141 phage show a ten-foldincrease, relative to the parental MA-BITI-E7, in the total fraction ofinput phage eluted from hNE-beads (0.3% vs 0.03%). The total fraction ofMA-BITI-E7-141 phage eluted from the hNE-beads is nearly twice that ofMA-EpiNE7 phage.

[0119] The above results show that binding by MA-BITI-E7-141 displayphage to hNE-beads is comparable to that of MA-EpiNE7 phage. If the twoproteins (EpiNE7 and BITI-E7-141) in solution have similar affinitiesfor hNE, then the affinity of the BITI-E7-141 protein for hNE is on theorder of 1 pM. Such an affinity is approximately 100-fold greater thanthat estimated above for the parental protein (BITI-E7) and is 10⁵ to10⁶ times as great as the affinity for hNE reported for the intact ITIprotein.

EXAMPLE 7 Mutagenesis of BITI-E7-141

[0120] BITI-E7-141 differs from ITI-D1 at nine positions (1, 2, 4, 15,16, 18, 19, 31, and 34). To obtain the protein having the fewest changesfrom ITI-D1 while retaining high specific affinity for hNE, we haveinvestigated the effects of reversing the changes at positions 1, 2, 4,16, 19, 31, and 34. The derivatives of BITI-E7-141 that were tested areMUT1619, MUTP1, and MUTT26A. The derivatives of BITI that were testedare AMINO1 and AMINO₂. The derivative of BITI-E7 that was tested isMUTQE. All of these sequences are shown in Table 100. MUT1619 restoresthe ITI-D1 residues Ala₁₆ and Ser₁₉. The sequence designated “MUTPl”asserts the amino acids I₁₅, G₁₆, S₁₉ in the context of BITI-E7-141. Itis likely that M₁₇ and F₁₈ are optimal for high affinity hNE binding.G₁₆ and S₁₉ occurred frequently in the high affinity hNE-bindingBPTI-variants obtained from fractionation of a library of BPTI-variantsagainst hNE (ROBE92). Three changes at the putative amino terminus ofthe displayed ITI-D1 domain were introduced to produce the MA-BITIseries of phage. AMINO1 carries the sequence K₁-E₂ while AMINO₂ carriesK₁-S₄. Other amino acids in the amino-terminal region of these sequencesare as in ITI-D1. MUTQE is derived from BITI-E7-141 by the alterationQ31E (reasseting the ITI-D1 w.t. residue). Finally, the mutagenicoligonucleotide MUTT26A is intended to remove a potential site ofN-linked glycosylation, N₂₄-G₂₅-T₂₆. In the intact ITI molecule isolatedfrom human serum, the light chain polypeptide is glycosylated at thissite (N₄₅, ODOM90). It is likely that N₂₄ will be glycosylated if theBITI-E7-141 protein is produced via eukaryotic expression. Suchglycosylation may render the protein immunogenic when used for long-termtreatment. The MUTT26A contains the alteration T26A and removes thepotential glycosylation site with minimal changes in the overallchemical properties of the residue at that position. In addition, an Alaresidue is frequently found in other BPTI homologues at position 26 (seeTable 34 of U.S. Pat. No. 5,223,409). Mutagenesis was performed on ssDNAof MA-BITI-E7-141 phage.

EXAMPLE 8 hNE-Binding Properties of Mutagenized MA-BITI-E7-141 DisplayPhage

[0121] Table 219 shows pH elution data for various display phage elutedfrom hNE-beads. Total pfu applied to the beads are in column two. Thefractions of this input pfu recovered in each pH fraction of theabbreviated pH elution protocol (pH 7.0, pH 3.5, and pH 2.0) are in thenext three columns. For data obtained using the extended pH elutionprotocol, the pH 3.5 listing represents the sum of the fractions ofinput recovered in the pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0, and pH3.5 elution samples. The pH 2.0 listing is the sum of the fractions ofinput obtained from the pH 3.0, pH 2.5, and pH 2.0 elution samples. Thetotal fraction of input pfu obtained throughout the pH elution protocolis in the sixth column. The final column of the table lists the totalfraction of input pfu recovered, normalized to the value obtained forMA-BITI-E7-141 phage.

[0122] Two factors must be considered when making comparisons among thedata shown in Table 219. The first is that due to the kinetic nature ofphage release from hNE-beads and the longer time involved in theextended pH elution protocol, the fraction of input pfu recovered in thepH 3.5 fraction will be enriched at the expense of the pH 2.0 fractionin the extended protocol relative to those values obtained in theabbreviated protocol. The magnitude of this effect can be seen bycomparing the results obtained when MA-BITI-E7-141 display phage wereeluted from hNE-beads using the two protocols. The second factor isthat, for the range of input pfu listed in Table 219, the input pfuinfluences recovery. The greater the input pfu, the greater the totalfraction of the input recovered in the elution. This effect is apparentwhen input pfu differ by more than a factor of about 3 to 4. The effectcan lead to an overestimate of affinity of display phage for hNE-beadswhen data from phage applied at higher titers is compared with that fromphage applied at lower titers.

[0123] With these caveats in mind, we can interpret the data in Table219. The effects of the mutations introduced into MA-BITI-E7-141 displayphage (“parental”) on binding of display phage to hNE-beads can begrouped into three categories: those changes that have little or nomeasurable effects, those that have moderate (2- to 3-fold) effects, andthose that have large (>5-fold) effects.

[0124] The MUTT26A and MUTQE changes appear to have little effect on thebinding of display phage to hNE-beads. In terms of total pfu recovered,the display phage containing these alterations bind as well as theparental to hNE-beads. Indeed, the pH elution profiles obtained for theparental and the MUTT26A display phage from the extended pH elutionprotocol are indistinguishable. The binding of the MUTTQE display phageappears to be slightly reduced relative to the parental and, in light ofthe applied pfu, it is likely that this binding is somewhatoverestimated.

[0125] The sequence alterations introduced via the MUTP1 and MUT1619oligonucleotides appear to reduce display phage binding to hNE-beadsabout 2- to 3-fold. In light of the input titers and the distributionsof pfu recovered among the various elution fractions, it is likelythat 1) both of these display phage have lower affinities for hNE-beadsthan do MA-EpiNE7 display phage, and 2) the MUT1619 display phage have agreater affinity for hNE-beads than do the MUTP1 display phage.

[0126] The sequence alterations at the amino terminus of BITI-E7-14appear to reduce binding by the display phage to hNE-beads at least tenfold. The AMINO2 changes are likely to reduce display phage binding to asubstantially greater extent than do the AMINO1 changes.

[0127] On the basis of the above interpretations of the data in Table219, we can conclude that:

[0128] 1.) The substitution of ALA for THR at position 26 in ITI-D1 andits derivatives has no effect on the interaction of the inhibitor withhNE. Thus, the possibility of glycosylation at Asn₂₄ of an inhibitorprotein produced in eukaryotic cell culture can be avoided with noreduction in affinity for hNE.

[0129] 2.) The increase in affinity of display phage for hNE-beads fromthe changes E31Q and Q34V results primarily from the Val substitution at34.

[0130] 3.) All three changes at the amino terminal region of ITI-D1(positions 1,2, and 4) influence display phage binding to hNE-beads tovarying extents. The S4F alteration seems to have about the same effectas does E2P. The change at position 1 appears to have only a smalleffect.

[0131] 4.) The changes in the region around the P1 residue inBITI-E7-141 (position 15) influence display phage binding to hNE. Thechanges A16G and P19S appear to reduce the affinity of the inhibitorsomewhat (perhaps 3-fold). The substitution of 115V further reducesbinding.

[0132] BITI-E7-141 differs from ITI-D1 at nine positions. From thediscussion above, it appears likely that a high affinity hNE-inhibitorbased on ITI-D1 could be constructed that would differ from the ITI-D1sequence at only five or six positions. These differences would be: Proat position 2, Phe at position 4, Val at position 15, Phe at position18, Val at position 34, and Ala at position 26. If glycosylation ofAsn₂₄ is not a concern Thr could be retained at 26.

[0133] Summary: Estimated Affinities of Isolated ITI-D1 Derivatives forhNE

[0134] On the basis of display phage binding to and elution from hNEbeads, it is possible to estimate affinities for hNE that variousderivatives of ITI-D1 may display free in solution. These estimates aresummarized in Table 55.

[0135] hNE Inhibitors Derived from ITI Domain 2

[0136] In addition to hNE inhibitors derived from ITI-D1, the presentinvention comprises hNE inhibitors derived from ITI-D2. These inhibitorshave been produced in Pichia pastoris in good yield. EPI-HNE-4 inhibitshuman neutrophil elastase with a K_(D)≈5 pM.

[0137] Purification and Properties of EPI-HNE Proteins

[0138] I. EPI-HNE Proteins.

EXAMPLE 9

[0139] Amino-acid sequences of EPI-HNE-3 and EPI-HNE-4 Table 100 givesamino acid sequences of four human-neutrophil-elastase (hNE) inhibitorproteins: EPI-HNE-1 (identical to EpiNEl), EPI-HNE-2, EPI-HNE-3, andEPI-HNE-4. These proteins have been derived from the parentalKunitz-type domains shown. Each of the proteins is shown aligned to theparental domain using the six cysteine residues (shaded) characteristicof the Kunitz-type domain. Residues within the inhibitor proteins thatdiffer from those in the parental protein are in upper case. Entireproteins having the sequences EPI-HNE-1, EPI-HNE-2, EPI-HNE-3, andEPI-HNE-4 (Table 100) have been produced. Larger proteins that compriseone of the hNE-inhibiting sequences are expected to have potenthNE-inhibitory activity; EPI-HNE-1, EPI-HNE-2, EPI-HNE-3, and EPI-HNE-4are particularly preferred. It is expected that proteins that comprise asignificant part of one of the hNE-inhibiting sequences found in Table100 (particularly if the sequence starting at or before the firstcysteine and continuing through or beyond the last cysteine is retained)will exhibit potent hNE-inhibitory activity.

[0140] The hNE-inhibitors EPI-HNE-1 and EPI-HNE-2 are derived from thebovine protein BPTI (aprotinin). Within the Kunitz-type domain, thesetwo inhibitors differ from BPTI at the same eight positions: K15I, R17F,118F, 119P, R39M, A40G, K41N, and R42G. In addition, EPI-HNE-2 differsfrom both BPTI and EPI-HNE-1 in the presence of four additional residues(EAEA) present at the amino terminus. These residues were added tofacilitate secretion of the protein in Pichia pastoris.

[0141] EPI-HNE-3 is derived from the second Kunitz domain of the lightchain of the human inter-α-trypsin inhibitor protein (ITI-D2). The aminoacid sequence of EPI-HNE-3 differs from that of ITI-D2(3-58) at onlyfour positions: R151,118F, Q19P and L20R. EPI-HNE-4 differs fromEPI-HNE-3 by the substitution A3E (the amino-terminal residue) whichboth facilitates secretion of the protein in P. pastoris and improvesthe K_(D) for hNE. Table 602 gives some physical properties of the hNEinhibitor proteins. All four proteins are small, high-affinity (K_(i)=2to 6 pM), fast-acting (k_(on)=4 to 11×10⁶ M⁻¹s⁻¹) inhibitors of hNE.

[0142] II. Production of the hNE-Inhibitors EPI-HNE-2, EPI-HNE-3, andEPI-HNE-4.

EXAMPLE 10 Pichia pastoris Production System.

[0143] Transformed strains of Pichia pastoris were used to express thevarious EPI-HNE proteins derived from BPTI and ITI-D2. Proteinexpression cassettes are cloned into the plasmid pHIL-D2 using the BstBIand EcoRI sites (Table 111). The DNA sequence of pHIL-D2 is given inTable 250. The cloned gene is under transcriptional control of P.pastoris upstream (labeled “aox1 5′”) aox1 gene promoter and regulatorysequences (dark shaded region) and downstream polyadenylation andtranscription termination sequences (second cross-hatched region,labeled “aox1 3′”). P. pastoris GS115 is a mutant strain containing anon-functional histidinol dehydrogenase (his4) gene. The his4 genecontained on plasmid pHIL-D2 and its derivatives can be used tocomplement the histidine deficiency in the host strain. Linearization ofplasmid pHIL-D2 at the indicated SacI site directs plasmid incorporationinto the host genome at the aox1 locus by homologous recombinationduring transformation. Strains of P. pastoris containing integratedcopies of the expression plasmid will express protein genes undercontrol of the aox1 promoter when the promoter is activated by growth inthe presence of methanol as the sole carbon source.

[0144] We have used this high density Pichia pastoris production systemto produce proteins by secretion into the cell CM. Expression plasmidswere constructed by ligating synthetic DNA sequences encoding the S.cerevisiae mating factor α prepro peptide fused directly to the aminoterminus of the desired hNE inhibitor into the plasmid pHIL-D2 using theBstBI and the EcoRI sites shown. Table 251 gives the DNA sequence of aBstBI-to-EcoRI insert that converts pHIL-D2 intopHIL-D2(MFa-PrePro::EPI-HNE-3). In this construction, the fusion proteinis placed under control of the upstream inducible P. pastoris aox1 genepromoter and the downstream aoxl gene transcription termination andpolyadenylation sequences. Expression plasmids were linearized by SacIdigestion and the linear DNA was incorporated by homologousrecombination into the genome of the P. pastoris strain GS115 byspheroplast transformation. Regenerated spheroplasts were selected forgrowth in the absence of added histidine, replated, and individualisolates were screened for methanol utilization phenotype (mut⁺),secretion levels, and gene dose (estimated via Southern hybridizationexperiments). High level secretion stains were selected for productionof hNE inhibitors: PEY-33 for production of EPI-HNE-2 and PEY-43 forproduction of EPI-HNE-3. In both of these strains, we estimate that fourcopies of the expression plasmid are integrated as a tandem array intothe aox1 gene locus.

[0145] To facilitate alteration of the Kunitz-domain encoding segment ofpHIL-D2 derived plasmids, we removed two restriction sites given inTable 111: the BstBI at 4780 and the AatII site at 5498. Thus, theKunitz-domain encoding segment is bounded by unique AatII and EcoRIsites. The new plasmids are called pD2pick(“insert”) where “insert”defines the domain encoded under control of the aox1 promoter. Table 253gives the DNA sequence of pD2pick(MFα::EPI-HNE-3). Table 254 gives alist of restriction sites in pD2pick(MFα::EPI-HNE-3).

[0146] EPI-HNE-4 is encoded by pD2pick(MFαPrePro::EPI-HNE-4) whichdiffers from pHIL-D2 in that: 1) the AatII/EcoRI segment of the sequencegiven in Table 251 is replaced by the segment shown in Table 252 and 2)the changes in the restriction sites discussed above have been made.Strain PEY-53 is P. pastoris GS115 transformed withpD2pick(MFα::EPI-HNE-4).

EXAMPLE 11 Protein Production

[0147] To produce the proteins, P. pastoris strains were grown inmixed-feed fermentations similar to the procedure described by Digan etal. (DIGA89). Although others have reported production of Kunitz domainsin P. pastoris, it is well known that many secretion systems involveproteases. Thus, it is not automatic that an altered Kunitz domainhaving a high potency in inhibiting hNE could be secreted from P.pastoris because the new inhibitor might inhibit some key enzyme in thesecretion pathway. Nevertheless, we have found that P. pastoris cansecrete hNE inhibitors in high yield.

[0148] Briefly, cultures were first grown in batch mode with glycerol asthe carbon source. Following exhaustion of glycerol, the culture wasgrown for about four hours in glycerol-limited feed mode to furtherincrease cell mass and to derepress the aoxl promoter. In the finalproduction phase, the culture was grown in methanol-limited feed mode.During this phase, the aox1 promoter is fully active and protein issecreted into the CM.

[0149] Table 607 and Table 608 give the kinetics of cell growth(estimated as A₆₀₀) and protein secretion (mg/l) for cultures of PEY-33and PEY-43 during the methanol-limited feed portions of the relevantfermentations. Concentrations of the inhibitor proteins in thefermentation cultures were determined from in vitro assays of hNEinhibition by diluted aliquots of cell-free culture media obtained atthe times indicated. Despite similarities in gene dose, fermentationconditions, cell densities, and secretion kinetics, the finalconcentrations of inhibitor proteins secreted by the two strains differby nearly an order of magnitude. The final concentration of EPI-HNE-2 inthe PEY-33 fermentation CM was 720 mg/l. The final concentration ofEPI-HNE-3 in the PEY-43 fermentation CM was 85 mg/l. The differences infinal secreted protein concentrations may result from idiosyncraticdifferences in the efficiencies with which the yeast synthesis andprocessing systems interact with the different protein sequences.

[0150] Strain PEY-33 secreted EPI-HNE-2 protein into the CM as a singlemolecular species which amino acid composition and N-terminal sequencingreveled to be the correctly-processed Kunitz domain with the sequenceshown in Table 601. The major molecular species produced by PEY-43cultures was the properly-processed EPI-HNE-3 protein. However, thisstrain also secreted a small amount (about 15% to 20% of the totalEPI-HNE-3) of incorrectly-processed material. This material proved to bea mixture of proteins with amino terminal extensions (primarily nine orseven residues in length) arising from incorrect cleavage of the MF aPrePro leader peptide from the mature Kunitz domain. The correctlyprocessed protein was purified substantially free of these contaminantsas described below.

[0151] III. Purification of the hNE-Inhibitors EPI-HNE-2 and EPI-HNE-3.

[0152] The proteins can be readily purified from fermenter CM bystandard biochemical techniques. The specific purification procedurevaries with the specific properties of each protein as described below.

EXAMPLE 12 Purification of EPI-HNE-2

[0153] Table 603 gives particulars of the purification of EPI-HNE-2,lot 1. The PEY-33 fermenter culture was harvested by centrifugation at8000× g for 15 min and the cell pellet was discarded. The 3.3 litersupernatant fraction was microfiltered used a Minitan UltrafiltrationSystem (Millipore Corporation, Bedford, Mass.) equipped with four 0.2μfilter packets.

[0154] The filtrate obtained from the microfiltration step was used intwo subsequent ultrafiltration steps. First, two 30K ultrafiltrationswere performed on the 0.2μ microfiltrate using the Minitan apparatusequipped with eight 30,000 NMWL polysulfone filter plates (#PLTKOMPO₄,Millipore Corporation, Bedford, Mass.). The retentate solution from thefirst 30K ultrafiltration was diluted with 10 mM NaCltrate, pH=3.5, andsubjected to a second 30K ultrafiltration. The two 30K ultrafiltrateswere combined to give a final volume of 5 liters containing about 1.4gram of EPI-HNE-2 protein (estimated from hNE-inhibition measurements).

[0155] The 30K ultrafiltrate was concentrated with change of buffer inthe second ultrafiltration step using the Minitan apparatus equippedwith eight 5,000 NMWL filter plates (#PLCCOMPO4, Millipore Corporation,Bedford, Mass.). At two times during the 5K ultrafiltration, theretentate solution was diluted from about 300 ml to 1.5 liters with 10mM NaCltrate, pH=3.5. The final 5K ultrafiltration retentate (Ca. 200ml) was diluted to a final volume of 1000 ml with 10 mM NaCltrate,pH-3.5.

[0156] EPI-HNE-2 protein was obtained from the 5K ultrafiltrationretentate solution by ammonium sulfate precipitation at RT. 100 ml of0.25 M ammonium acetate, pH=3.2, ({fraction (1/10)} volume) was added tothe 5K ultrafiltration retentate, followed by one final volume (1.1liter) of 3 M ammonium sulfate. Following a 30 minute incubation at RT,precipitated material was pelleted by centrifugation at 10,000× g for 45minutes. The supernatant solution was removed, the pellet was dissolvedin water in a final volume of 400 ml, and the ammonium sulfateprecipitation was repeated using the ratios described above. The pelletfrom the second ammonium sulfate precipitation was dissolved in 50 mMsodium acetate, pH=3.5 at a final volume of 300 ml.

[0157] Residual ammonium sulfate was removed from the EPI-HNE-2preparation by ion exchange chromatography. The solution from theammonium sulfate precipitation step was applied to a strongcation-exchange column (50 ml bed volume Macroprep 50S) (Bio-RadLaboratories, Inc, Hercules, Calif.) previously equilibrated with 10 mMNaCltrate, pH=3.5. After loading, the column was washed with 300 ml of10 mM NaCltrate, pH=3.5. EPI-HNE-2 was then batch-eluted from the columnwith 300 ml of 50 mM NH₄OAc, pH=6.2. Fractions containing EPI-HNE-2activity were pooled and the resulting solution was lyophilized. Thedried protein powder was dissolved in 50 ml dH₂O and the solution waspassed through a 0.2μ filter (#4192, Gelman Sciences, Ann Arbor, Mich.).The concentration of the active inhibitor in the final stock solutionwas determined to be 2 mM (13.5 mg/ml). This stock solution (EPI-HNE-2,Lot 1) has been stored as 1 ml aliquots at 4° C. and −70° C. for morethan 11 months with no loss in activity.

[0158] Table 603 summarizes the yields and relative purity of EPI-HNE-2at various steps in the purification procedure. The overall yield of thepurification procedure was about 30%. The major source of loss wasretention of material in the retentate fractions of the 0.2μmicrofiltration and 30k ultrafiltration steps.

EXAMPLE 13 Purification of EPI-HNE-3

[0159] Purification of EPI-HNE-3, lot 1, is set out in Table 604. ThePEY-43 fermenter culture was harvested by centrifugation at 8,000× g for15 min and the cell pellet was discarded. The supernatant solution (3100ml) was microfiltered through 0.2μ Minitan packets (4 packets). Afterthe concentration, a diafiltration of the retentate was performed sothat the final filtrate volume from the 0.2μ filtration was 3300 ml.

[0160] A 30K ultrafiltration was performed on the filtrate from the 0.2μmicrofiltration step. When the retentate volume had been reduced to 250ml, a diafiltration of the retentate was performed at a constantretentate volume (250 ml) for 30 min at a rate of 10 ml/min. Theresulting final volume of filtrate was 3260 ml.

[0161] EPI-HNE-3 protein and other medium components were found toprecipitate from solution when the fermenter CM was concentrated. Forthis reason, the 5 k ultrafiltration step was not performed.

[0162] Properly processed EPI-HNE-3 was purified substantially free ofmis-processed forms and other fermenter culture components by ionexchange chromatography. A 30 ml bed volume strong cation ion exchangecolumn (Macroprep 50S) was equilibrated with 10 mM NaCltrate pH=3.5. The30K ultrafiltration filtrate was applied to the column and binding ofEPI-HNE-3 to the column was confirmed by demonstrating the complete lossof inhibitor activity in the column flow through. The column was thenwashed with 300 ml of 10 mM NaCltrate, pH=3.5.

[0163] To remove EPI-HNE-3 from the column, we sequentially eluted itwith 300 ml volumes of the following solutions:

[0164] 100 mM ammonium acetate, pH=3.5

[0165] 50 mM ammonium acetate, pH=4.8

[0166] 50 mM ammonium acetate, pH=6.0

[0167] 50 mM ammonium acetate, pH=6.0, 0.1 M NaCl

[0168] 50 mM ammonium acetate, pH=6.0, 0.2 M NaCl

[0169] 50 mM ammonium acetate, pH=6.0, 0.3 M NaCl

[0170] 50 mM ammonium acetate, pH=6.0, 0.4 M NaCl

[0171] 50 mN Tris/Cl pH=8.0, 1.0 NaCl

[0172] The majority of the EPI-HNE-3 eluted in two 50 mM ammoniumacetate, pH=6.0 fractions. The 0.1 M NaCl fraction contained about 19%of the input EPI-HNE-3 activity (28 mg of 159 mg input) and essentiallyall of the mis-processed forms of EPI-HNE-3. The 0.2M NaCl fractioncontained about 72% (114 mg) of the input EPI-HNE-3 and was almostcompletely free of the higher molecular weight mis-processed forms andother UV-absorbing contaminants. The fractions from the 50 mM ammoniumacetate, pH=6.0, 0.2 M NaCl elution having the highest concentrations ofEPI-HNE-3 were combined (95 mg).

[0173] An ammonium sulfate precipitation was performed on the 0.2 MNaCl, pH=6.0 ion exchange column eluate. 800 ml of 3 M ammonium sulfatewas added to 160 ml of eluate solution (final ammonium sulfateconcentration=2.5 M) and the mixture was incubated at RT for 18 hours.The precipitated material was then pelleted by centrifugation at 10,000x g for 45 minutes. The supernatant fluid was discarded and the pelletedmaterial was dissolved in 100 ml of water.

[0174] Residual ammonium sulfate was removed from the EPI-HNE-3preparation by batch ion exchange chromatography. The pH of the proteinsolution was adjusted to 3.0 with diluted ({fraction (1/10)}) HOAc andthe solution was then applied to a 10 ml bed volume Macroprep 50S columnthat had been equilibrated with 10 mM NaCltrate, pH=3.5. Followingsample loading, the column was washed with 100 ml of 10 mM NaCltrate,pH=3.5 followed by 100 ml of dH₂O. EPI-HNE-3 was eluted from the columnwith 100 ml of 50 mM NH₄CO₃, pH=9.0. pH 9 fractions having the highestconcentrations of EPI-HNE-3 were combined (60 mg) and stored at 4° C.for 7 days before lyophilization.

[0175] The lyophilized protein powder was dissolved in 26 ml dH₂O andthe solution was passed through a 0.2p filter (#4912, Gelman Sciences,Ann Arbor, Mich.). The concentration of active inhibitor in the finalstock solution was found to be 250 μM (1.5 mg/ml). This stock solution(EPI-HNE-3, Lot 1) has been stored as 1 ml aliquots at −70° C. for morethan six months with no loss of activity. EPI-HNE-3 stored in watersolution (without any buffering) deteriorated when kept at 4° C. Afterfive months, about 70% of the material was active with a K_(i) of about12 pM. Table 604 gives the yield and relative purity of EPI-HNE-3 atvarious steps in the purification procedure. A major purification stepoccurred at the first ion exchange chromatography procedure. Theammonium sulfate precipitation step provided only a small degree offurther purification. Some loss of inhibitor activity occurred onincubation at pH=9 (See pH stability data). The production andpurification of EPI-HNE-1 and EPI-HNE-4 were analogous to that ofEPI-HNE-2 and EPI-HNE-3.

EXAMPLE 14 Tricine-PAGE Analysis of EPI-HNE-2 and EPI-HNE-3

[0176] The high resolution tricine gel system of Schagger and von Jagow(SCHA87) was used to analyze the purified proteins produced (videsupra). For good resolution of the low molecular weight EPI-HNE proteinswe used a 16.5% resolving layer with 10% separating and 4% stackinglayers. Following silver staining, we inspected a gel having:

[0177] Lane 1: EPI-HNE-2 25 ng,

[0178] Lane 2: EPI-HNE-2 50 ng,

[0179] Lane 3: EPI-HNE-2 100 ng,

[0180] Lane 4: EPI-HNE-2 200 ng,

[0181] Lane 5: EPI-HNE-3 25 ng,

[0182] Lane 6: EPI-HNE-3 50 ng,

[0183] Lane 7: EPI-HNE-3 100 ng,

[0184] Lane 8: EPI-HNE-3 200 ng, and

[0185] Lane 9: Molecular-weight standards: RPN 755, (AmershamCorporation, Arlington Heights, Ill.).

[0186] Stained proteins visible on the gel and their molecular weightsin Daltons are: ovalbumin (46,000), carbonic anhydrase (30,000), trypsininhibitor (21,500), lysozyme (14,300), and aprotinin (6,500). The amountof protein loaded was determined from measurements of hNE-inhibition. Wefound the following features. EPI-HNE-2, Lot 1 shows a single stainingband of the anticipated size (ca. 6,700 D) at all loadings. Similarly,EPI-HNE-3, Lot 1 protein shows a single staining band of the anticipatedsize (ca. 6,200 D). At the highest loading, traces of the highermolecular weight (ca. 7,100 D) incorrectly processed form can bedetected. On the basis of silver-stained high-resolution PAGE analysis,the purity of both protein preparations is assessed to be significantlygreater than 95%.

[0187] IV. Properties of EPI-HNE-2 and EPI-HNE-3.

[0188] A. Inhibition of hNE.

EXAMPLE 15 Measured K_(D)s of EPI-HNE Proteins for hNE

[0189] Inhibition constants for the proteins reacting with hNE (K_(i))were determined using RT measurements of hydrolysis of a fluorogenicsubstrate (N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin,Sigma M-9771) by hNE. For these measurements, aliquots of theappropriately diluted inhibitor stocks were added to 2 ml solutions of0.847 nM hNE in reaction buffer (50 mM Tris-Cl, pH=8.0, 150 mM NaCl, 1mM CaCl₂, 0.25% Triton-X-100) in plastic fluorescence cuvettes. Thereactions were incubated at RT for 30 minutes. At the end of theincubation period, the fluorogenic substrate was added at aconcentration of 25 μM and the time course for increase in fluorescenceat 470 nm (excitation at 380 nm) due to enzymatic substrate cleavage wasrecorded using a spectrofluorimeter (Perkin-Elmer 650-15) and stripchart recorder. We did not correct for competition between substrate andinhibitor because (S₀/K_(m)) is 0.07 (where S₀ is the substrateconcentration and K_(m) is the binding constant of the substrate forhNE). K_(i) is related to K_(apparent) byK_(i)=K_(apparent)×(1/(1+(S₀/K_(m)))). The correction is small comparedto the possible errors in K_(apparent). Rates of enzymatic substratecleavage were determined from the linear slopes of the recordedincreases in fluorescence. The percent residual activity of hNE in thepresence of the inhibitor was calculated as the percentage of the rateof fluorescence increase observed in the presence of the inhibitor tothat observed when no added inhibitor was present.

[0190] We recorded data used to determine K_(i) for EPI-HNE-2 andEPI-HNE-3 reacting with hNE. Data obtained as described above arerecorded as percent residual activity plotted as a function of addedinhibitor. Values for K_(i) and for active inhibitor concentration inthe stock are obtained from a least-squares fit program. From the data,K_(i) values for EPI-HNE-2 and for EPI-HNE-3 reacting with hNE at RTwere calculated to be 4.8 pM and 6.2 pM, respectively. Determinations ofK_(i) for EPI-HNE-2 and EPI-HNE-3 reacting with hNE are given in Table610 and Table 611.

[0191] The kinetic on-rates for the inhibitors reacting with hNE(k_(on)) were determined from measurements of progressive inhibition ofsubstrate hydrolytic activity by hNE following addition of inhibitor.For these experiments, a known concentration of inhibitor was added to asolution of hNE (0.847 nM) and substrate (25 μM) in 2 ml of reactionbuffer in a plastic fluorescence cuvette. The change in fluorescence wasrecorded continuously following addition of the inhibitor. In theseexperiments, sample fluorescence did not increase linearly with time.Instead, the rate of fluorescence steadily decreased reflectingincreasing inhibition of hNE by the added inhibitor. The enzymatic rateat selected times following addition of the inhibitor was determinedfrom the slope of the tangent to the fluorescence time course at thattime.

[0192] The kinetic constant k_(on) for EPI-HNE-2 reacting with hNE wasdetermined as follows. EPI-HNE-2 at 1.3 nM was added to buffercontaining 0.867 nM hNE (I:E=1.5:1) at time 0. Measured percent residualactivity was recorded as a function of time after addition of inhibitor.A least-squares fit program was used to obtain the value ofk_(on)=4.0×10⁶ M⁻¹s⁻¹.

[0193] The kinetic off rate, k_(off), is calculated from the measuredvalues of K_(i) and k_(on) as:

k _(off) =K _(D) ×k _(on).

[0194] The values from such measurements are included in Table 602. TheEPI-HNE proteins are small, high affinity, fast acting inhibitors ofhNE.

[0195] B. Specificity.

EXAMPLE 16 Specificity of EPI-HNE Proteins

[0196] We attempted to determine inhibition constants for EPI-HNEproteins reacting with several serine proteases. The results aresummarized in Table 605. In all cases except chymotrypsin, we wereunable to observe any inhibition even when 10 to 100 μM inhibitor wasadded to enzyme at concentrations in the nM range. In Table 605, ourcalculated values for K_(i) (for the enzymes other than chymotrypsin)are based on the conservative assumption of less than 10% inhibition atthe highest concentrations of inhibitor tested. For chymotrypsin, theK_(i) is about 10 μM and is probably not specific.

[0197] C. In Vitro Stability.

EXAMPLE 17 Resistance to Oxidative Inactivation.

[0198] Table 620 shows measurements of the susceptibility of EPI-HNEproteins to oxidative inactivation as compared with that of two othernatural protein hNE inhibitors: α1 Protease Inhibitor (API) andSecretory Leucocyte Protease Inhibitor (SLPI). API (10 μM), SLPI (8.5uM), EPI-HNE-1 (5 μM), EPI-HNE-2 (10 μM), EPI-HNE-3 (10 μM), andEPI-HNE-4 (10 μM) were exposed to the potent oxidizing agent,Chloramine-T, at the indicated oxidant:inhibitor ratios in 50 mMphosphate buffer, pH=7.0 for 20 minutes at RT. At the end of theincubation period, the oxidation reactions were quenched by addingmethionine to a final concentration of 4 mM. After a further 10 minuteincubation, the quenched reactions were diluted and assayed for residualinhibitor activity in our standard hNE-inhibition assay.

[0199] Both API and SLPI are inactivated by low molar ratios of oxidantto inhibitor. The Chloramine-T:protein molar ratios required for 50%inhibition of API and SLPI are about 1:1 and 2:1, respectively. Theseratios correspond well with the reported presence of two and fourreadily oxidized methionine residues in API and SLPI, respectively. Incontrast, all four EPI-HNE proteins retain essentially completehNE-inhibition activity following exposure to Chloramine-T at all molarratios tested (up to 50:1, in the cases of EPI-HNE-3 and EPI-HNE-4).Neither EPI-HNE-3 nor EPI-HNE-4 contain any methionine residues. Incontrast, EPI-HNE-1 and EPI-HNE-2 each contains two methionine residues(see Table 100). The resistance of these proteins to oxidativeinactivation indicates that the methionine residues are eitherinaccessible to the oxidant or are located in a region of the proteinthat does not interact with hNE.

EXAMPLE 18 pH Stability

[0200] Table 612 shows the results of measurements of the pH stabilityof EPI-HNE proteins. The stability of the proteins to exposure to pHconditions in the range of pH 1 to pH 10 was assessed by maintaining theinhibitors in buffers of defined pH at 37° C. for 18 hours anddetermining the residual hNE inhibitory activity in the standardhNE-inhibition assay. Proteins were incubated at a concentration of 1μM. The buffers shown in Table 14 were formulated as described (STOL90)and used in the pH ranges indicated: TABLE 14 Buffers used in stabilitystudies Buffer Lowest pH Highest pH Glycine-HCl 1 2.99 Citrate-Phosphate3 7 Phosphate 7 8 Glycine-NaOH 8.5 10

[0201] Both BPTI-derived inhibitors, EPI-HNE-1 and EPI-HNE-2, are stableat all pH values tested. EPI-HNE-3 and EPI-HNE-4, the inhibitors derivedfrom the human protein Kunitz-type domain, were stable when incubated atlow pH, but showed some loss of activity at high pH. When incubated at37° C. for 18 hours at pH=7.5, the EPI-HNE-3 preparation lost 10 to 15%of its hNE-inhibition activity. EPI-HNE-4 retains almost full activityto pH 8.5. Activity of the ITI-D2-derived inhibitor declined sharply athigher pH levels so that at pH 10 only 30% of the original activityremained. The sensitivity of EPI-HNE-3 to incubation at high pH probablyexplains the loss of activity of the protein in the final purificationstep noted previously.

EXAMPLE 19 Temperature Stability

[0202] The stability of EPI-HNE proteins to temperatures in the range 0°C. to 95° C. was assessed by incubating the inhibitors for thirtyminutes at various temperatures and determining residual inhibitoryactivity for hNE. In these experiments, protein concentrations were 1 1Min phosphate buffer at pH=7. As is shown in Table 630, the fourinhibitors are quite temperature stable.

[0203] EPI-HNE-1 and EPI-HNE-2 maintain full activity at alltemperatures below about 90° C. EPI-HNE-3 and EPI-HNE-4 maintain fullinhibitory activity when incubated at temperatures below 65° C. Theactivity of the protein declines somewhat at higher temperatures.However, all three proteins retain more than ≈50% activity even whenincubated at 95° C. for 30 minutes.

EXAMPLE 20 Routes to Other hNE-Inhibitory Sequences

[0204] The present invention demonstrates that very high-affinity hNEinhibitors can be devised from Kunitz domains of human origin with veryfew amino-acid substitutions. It is believed that almost any Kunitzdomain can be made into a potent and specific hNE inhibitor with eightor fewer substitutions. In particular, any one of the known human Kunitzdomains could be remodeled to provide a highly stable, highly potent,and highly selective hNE inhibitor. There are at least three routes tohNE inhibitory Kunitz domains: 1) replacement of segments known to beinvolved in specifying hNE binding, 2) replacement of single residuesthought to be important for hNE binding, and 3) use of libraries ofKunitz domains to select hNE inhibitors.

EXAMPLE 21 Substitution of Segments in Kunitz Domains

[0205] Table 100 shows the amino-acid sequences of 11 human Kunitzdomains. These sequences have been broken into ten segments: 1:Nterminus-residue 4; 2:residue 5; 3:6-9(or 9a); 4:10-13; 5:14; 6:15-21;7:22-30, 8:31-36; 8:37-38; 9:39-42; and 10:43-C terminus (or 42a-Cterminus).

[0206] Segments 1, 3, 5, 7, and 9 contain residues that stronglyinfluence the binding properties of Kunitz domains and are doubleunderscored in the Consensus Kunitz Domain of Table 100. Other thansegment 1, all the segments are the same length except for TFPI-2 Domain2 which carries an extra residue in segment 2 and two extra residues insegment 10.

[0207] Segment 1 is at the amino terminus and influences the binding byaffecting the stability and dynamics of the protein. Segments 3, 5, 7,and 9 contain residues that contact serine proteases when a Kunitzdomain binds in the active site. High-affinity hNE inhibition requires amolecule that is highly complementary to hNE. Segments 3, 5, 7, and 9supply the amino acids that contact the protease. The sequences insegments 1, 3, 5, 7, and 9 must work together in the context supplied byeach other and the other segments. Nevertheless, we have demonstratedthat very many different sequences are capable of high-affinity hNEinhibition.

[0208] It may be desirable to have an hNE inhibitor that is highlysimilar to a human protein to reduce the chance of immunogenicity.Candidate high-affinity hNE inhibitor protein sequences may be obtainedby taking an aprotonin-type Kunitz domain that strongly or very stronglyinhibits hNE, and replacing one, two, three, four or all of segments 2,4, 6, 8, and 10 with the corresponding segment from a human Kunitzdomain, such as those listed in Table 100, or other domain known to haverelatively low immunogenicity in humans. (Each of segments 2, 4, 6, 8,and 10 may be taken from the same human domain, or they may be takenfrom different human domains.) Alternatively, a reduced immunogenicity,high hNE inhibiting domain may be obtained by taking one of the humanaprotonin-type Kunitz domains and replacing one, two, three or all ofsegments 3, 5, 7 and 9 (and preferably also segment 1) with thecorresponding segment from one or more aprotonin-like Kunitz domainsthat strongly or very strongly inhibit hNE. In making these humanizedhNE inhibitors, one may, of course, use, rather than a segment identicalto that of one of the aforementioned source proteins, a segment whichdiffers from the native source segment by one or more conservativemodifications. Such differences should, of course, be taken with dueconsideration for their possible effect on inhibitory activity and/orimmunogenicity. In some cases, it may be advantageous that the segmentbe a hybrid of corresponding segments from two or more human domains (inthe case of segments 2, 4, 6, 8 and 10) or from two or more strong orvery strong hNE inhibitor domains (in the case of segments 3, 5, 7, and9). Segment 1 may correspond to the segment 1 of a strong or very stronghNE inhibitor, or the segment 1 of a human aprotonin-like Kunitz domain,or be a chimera of segment l's from both.

[0209] The proteins DPI.1.1, DPI.2.1, DPI.3.1, DPI.4.1, DPI.5.1,DPI.6.3, DPI.7.1, DPI.8.1, and DPI.9.1 were designed in this way.DPI.1.1 is derived from App-I by replacing segments 3, 5, 7, and 9 withthe corresponding segments from EPI-HNE-1. DPI.2.1 is derived fromTFPI2-D1 by replacing segments 3, 5, 7, and 9 with the correspondingresidues from EPI-HNE-1. DPI.3.1 is derived from TFPI2-D2 by replacingresidues 9a-21 with residues 10-21 of EPI-HNE-4 and replacing residues31-42b with residues 31-42 of EPI-HNE-4. DPI.4.1 is derived fromTFPI2-D3 by replacing segments 3, 5, 7, and 9 with the correspondingresidues from MUTQE. DPI.5.1 is derived from LACI-D1 by replacingsegments 3, 5, 7, and 9 with the corresponding residues from MUTQE.DPI.6.1 is derived from LACI-D2 by replacing segments 3, 5, 7, and 9with the corresponding residues from MUTQE. DPI.7.1 is derived fromLACI-D3 by replacing segments 3, 5, 7, 9 with the corresponding residuesfrom EPI-HNE-4. DPI.8.1 is derived from the A3 collogen Kunitz domain bysubstitution of segments 3, 5, 7, and 9 from EPI-HNE-4. DPI.9.1 isderived from the HKI B9 domain by replacing segments 3, 5, 7, and 9 withthe corresponding residues from EPI-HNE-4.

[0210] While the above-described chimera constitute preferredembodiments of the present invention, the invention is not limited tothese chimera.

EXAMPLE 22 Point substitutions in Kunitz Domains

[0211] In this example, certain substitution mutations are discussed. Itmust be emphasized that this example describes preferred embodiments ofthe invention, and is not intended to limit the invention.

[0212] All of the protein sequences mentioned in this example are to befound in Table 100. Designed protease inhibitors are designated “DPI”and are derived from human Kunitz domains (also listed in Table 100).Each of the sequences designated DPI.i.2 (for i=1 to 9) is derived fromthe domain two above it in the table by making minimal point mutations.Each of the sequences designated DPI.i.3 (for i =1 to 9) is derived fromthe sequence three above it by more extensive mutations intended toincrease affinity. For some parental domains, additional examples aregiven. The sequences designated DPI.i.1 are discussed in Example 21.

[0213] The most important positions are 18 and 15. Any Kunitz domain islikely to become a good hNE inhibitor if Val or Ile is at 15 (with Ilebeing preferred) and Phe is at 18. (However, these features are notnecessarily required for such activity.)

[0214] If a Kunitz domain has Phe at 18 and either Ile or Val at 15 andis not a good hNE inhibitor, there may be one or more residues in theinterface preventing proper binding.

[0215] The Kunitz domains having very high affinity for hNE hereindisclosed (as listed in Table 100) have no charged groups at residues10, 12 through 19, 21, and 32 through 42. At position 11, only neutraland positively charged groups have been observed in very high affinityhNE inhibitors. At position 31, only neutral and negatively chargedgroups have been observed in high-affinity hNE inhibitors. If a parentalKunitz domain has a charged group at any of those positions where onlyneutral groups have been observed, then each of the charged groups ispreferably changed to an uncharged group picked from the possibilitiesin Table 790 as the next step in improving binding to hNE. Similarly,negatively charged groups at 11 and 19 and positively charged groups at31 are preferably replaced by groups picked from Table 790.

[0216] At position 10, Tyr, Ser, and Val are seen in high-affinity hNEinhibitors. Asn or Ala may be allowed since this position may notcontact hNE. At position 11, Thr, Ala, and Arg have been seen inhigh-affinity hNE inhibitors. Gin and Pro are very common at 11 inKunitz domains and may be acceptable. Position 12 is almost always Gly.If 12 is not Gly, try changing it to Gly.

[0217] All of the high-affinity hNE inhibitors produced so far havePro₁₃, but it has not been shown that this is required. Many (62.5%)Kunitz domains have Pro₁₃. If 13 is not Pro, then changing to Pro mayimprove the hNE affinity. Val, Ala, Leu, or Ile may also be acceptablehere.

[0218] Position 14 is Cys. It is possible to make domains highly similarto Kunitz domains in which the 14-38 disulfide is omitted. Such domainsare likely to be less stable than true Kunitz domains having the threestandard disulfides.

[0219] Position 15 is preferably Ile or Val. Ile is more preferred.

[0220] Most Kunitz domains (82%) have either Gly or Ala at 16 and thismay be quite important. If residue 16 is not Gly or Ala, change 16 toeither Gly or Ala; Ala is preferred. Position 17 in very potent hNEinhibitors has either Phe or Met; those having Ile or Leu at 17 are lesspotent. Phe is preferred. Met should be used only if resistance tooxidation is not important. Position 18 is Phe.

[0221] It has been shown that high-affinity hNE inhibitors may haveeither Pro or Ser at position 19. Gln or Lys at position 19 may beallowed. At position 21, Tyr and Trp have been seen in very highaffinity hNE inhibitors; Phe may also work.

[0222] At position 31, Gin, Glu, and Val have been observed in highaffinity hNE inhibitors. Since this is on the edge of the bindinginterface, other types are likely to work well. One should avoid basictypes (Arg and Lys). At position 32, Thr and Leu have been observed inhigh-affinity hNE inhibitors. This residue may not make direct contactand other uncharged types may work well. Pro is very common here. Serhas been seen and is similar to Thr. Ala has been seen in natural Kunitzdomains and is unlikely to make any conflict. Position 33 is always Phein Kunitz domains.

[0223] It appears that many amino acid types may be placed at position34 while retaining high affinity for hNE; large hydrophobic residues(Phe, Trp, Tyr) are unfavorable. Val and Pro are most preferred at 34.Positions 35-38 contain the sequence Tyr-Gly-Gly-Cys. There is a littlediversity at position 36 in natural Kunitz domains. In the BPTI-Trypsincomplex, changing Gly₃₆ to Ser greatly reduces the binding to trypsin.Nevertheless, S36 or T36 may not interfere with binding to hNE and couldeven improve it. If residue 36 is not Gly, one should consider changingit to Gly.

[0224] Position 39 seems to tolerate a variety of types. Met and Gln areknown to work in very high-affinity inhibitors. Either Ala or Gly areacceptable at position 40; Gly is preferred. At position 41, Asn is byfar the most common type in natural Kunitz domains and may act tostabilize the domains. At position 42, Gly is preferred, but Ala isallowed.

[0225] Finally, positions that are highly conserved in Kunitz domainsmay be converted to the conserved type if needed. For example, themutations X36G, X37G, X41N, and X12G may be desirable in those casesthat do not already have these amino acids at these positions.

[0226] The above mutations are summarized in Table 711. Table 711contains, for example, mutations of the form X15I which means change theresidue at position 15 (whatever it is) to Ile or leave it alone if itis already Ile. A Kunitz domain that contains the mutation X18F andeither X15I or X15V (X15I preferred) will have strong affinity for hNE.As from one up to about 8 of the mutations found in Table 711 areasserted, the affinity of the protein for hNE will increase so that theKi approaches the range 1-5 pM.

[0227] The sequence DPI.1.2 was constructed from the sequence of App-Iby the changes R151,118F, and F34V and should be a potent hNE inhibitor.DPI.1.3 is likely to be a more potent inhibitor, having the changesR15I, M17F (to avoid sensitivity to oxidation), 118F, P32T, F34V, andG39M.

[0228] DPI.2.2 was derived from the sequence of TFPI2-D1 by the changesR15I, L18F, and L34V and should be a potent hNE inhibitor. DPI.2.3 maybe more potent due to the changes Y11T, R15I, L17F, L18F, R31Q, Q32T,L34V, and E39M. DPI.3.2 is derived from TFPI2-D2 by the changes E15I,T18F, S26A(to prevent glycosylation), K32T, and F34V and should be apotent hNE inhibitor. DPI.3.3 may be more potent by having the changesΔ9a, D11A, D12G, Q13P, E15I, S17F, T18F, E19K, K20R, N24A (to preventglycosylation), K32T, F34V, and A42a-42b.

[0229] DPI.4.2 is derived from TFPI2-D3 by the changes S15I, N17F, andV18F and should be a potent inhibitor of hNE. DPI.4.3 may be more potentby having the changes E11T, L13P, S15I, N17F, V18F, A32T, T34V, andT36G.

[0230] DPI.5.2 is derived from LACI-D1 by the changes K15I and M18F andis likely to be a potent inhibitor of hNE. DPI.5.3 may be more potentdue to the changes D10Y, D11T, K15I, 117F, M18F, and E32T. Other changesthat may improve DPI.5.3 include F21W, 134V, E39M, and Q42G.

[0231] The sequence of DPI.6.2 was constructed from the sequence ofhuman LACI-D2 by the mutations R15V and 118F. The rest of the sequenceof LACI-D2 appears to be compatible with hNE binding. DPI.6.3 carriestwo further mutations that make it more like the hNE inhibitors heredisclosed: Y17F and K34V. Other alterations that are likely to improvethe hNE binding of LACI-D2 include 113P, R32T, and D10S. DPI.6.4 isderived from DPI.6.3 by the additional alteration N25A that will preventglycosylation when the protein is produced in a eukaryotic cell. Othersubstitutions that would prevent glycosylation include: N25K, T27A,T27E, N25S, and N25S. DPI.6.5 moves further toward the ITI-D1, ITI-D2,and BPTI derivatives that are known to have affinity for hNE in the 1-5pM range through the mutations 113P, R15V, Y17F, 118F, T19Q, N25A, K34V,and L39Q. In DPI.6.6, the T19Q and N25A mutations have been reverted.Thus the protein would be glycosylated in yeast or other eukaryoticcells at N₂₅. DPI.6.7 carries the alterations 113P, R15I, Y17F, 118F,T19P, K34V, and L39Q.

[0232] DPI.7.2 is derived from human LACI domain 3 by the mutations R15Vand E18F. DPI.7.3 carries the mutations R15V, N17F, E18F, and T46K. TheT46K mutation should prevent glycosylation at N₄₄. DPI.7.4 carries moremutations so that it is much more similar to the known high-affinity hNEinhibitors. The mutations are D10V, L13P, R15V, N17F, E18F, K34V, S36G,and T46K. DPI.7.5 carries a different set of alterations: L13P, R15I,N17F, E18F, N19P, F21W, R31Q, P32T, K34V, S36G, and T46K; DPI.7.5 shouldnot be glycosylated in eukaryotic cells.

[0233] DPI.8.2 is derived from the sequence of the A3 collagen Kunitzdomain by the changes R15I, D16A, 118F, and W34V and is expected to be apotent hNE inhibitor. DPI.8.3 is derived from the A3 collagen Kunitzdomain by the changes T13P, R15I, D16A, 118F, K20R, and W34V.

[0234] DPI.9.2 is derived from the HKI B9 Kunitz domain by the changesQ15I, T16A, and M18F and is expected to be a potent hNE inhibitor.DPI.9.3 may be more potent due to the changes Q15I, T16A, M18F, T19P,E31V, and A34V.

EXAMPLE 23 Libraries of Kunitz Domains

[0235] Other Kunitz domains that can potently inhibit hNE may be derivedfrom human Kunitz domains either by substituting hNE-inhibitingsequences into human domains or by using the methods of US 5,223,409 andrelated patents. Table 720 shows a gene that will cause display of humanLACI-D2 on M13 gIIIp; essentially the same gene could be used to achievedisplay on M13 gVIIIp or other anchor proteins (such as bacterialouter-surface proteins (OSPs)). Table 725 shows a gene to cause displayof human LACI D1.

[0236] Table 730 and Table 735 give variegations of LACI-D1 and LACI-D2respectively. Each of these is divided into variegation of residues10-21 in one segment and residues 31-42 in another. In each case, theappropriate vgDNA is introduced into a vector that displays the parentalprotein and the library of display phage are fractionated for binding toimmobilized hNE. TABLE 13 BPTI Homologues (1-20) R # 1 2 3 4 5 6 7 8 910 11 12 13 14 15 16 17 18 19 20  −5 —  −4 —  −3 — — — F — — — — — — — —— — — — Z — — —  −2 — — — Q T — — — — — — Q — — — H G Z — Z  −1 — — — TE — — — — — — P — — — D D G — P    1 R R R P R R R R R R R L A R R R K RA R    2 P P P P P P P P P P P R A P P P R P A R    3 D D D D D D D D DD D K K D R T D S K K    4 F F F L F F F F F F F L Y F F F I F Y L    5C C C C C C C C C C C C C C C C C C C C    6 L L L Q L L L L L L L I K EE N R N K I    7 E E E L E E E E E E E L L L L L L L L L    8 P P P P PP P P P P P H P P P P P P P H    9 P P P Q P P P P P P P R L A A P P A VR   10 Y Y Y A Y Y Y Y Y Y Y N R E E E E E R N   11 T T T R T T T T T TT P I T T S Q T Y P   12 G G G G G G G G G G G G G G G G G G G G   13 PP P P P P P P P P P R P L L R P P P R   14 C T A C C C C C C C C C C C CC C C C C   15 K K K K K V G A L I K Y K K K R K K K Y   16 A A A A A AA A A A A Q R A A G G A K D   17 R R R A A R R R R R R K K Y R H R S K K  18 I I I L M I I I I I I I I I I I L I F I   19 I I I L I I I I I I IP P R R R P R P P   20 R R R R R R R R R R R A S S S R R Q S A   21 Y YY Y Y Y Y Y Y Y Y F F F F I Y Y F F   22 F F F F F F F F F F F Y Y H H YF Y Y Y   23 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y   24 N N N N N N NN N N N N K N N N N N N N   25 A A A S A A A A A A A Q W L R L P S W Q  26 K K K T K K K K K K K K K A A E A K K K   27 A A A S A A A A A A AK A A A S S S A K   28 G G G N G G G G G G G K K Q Q N R G K K   29 L LL A F L L L L L L Q Q Q Q K M G Q Q   30 C C C C C C C C C C C C C C C CC C C C   31 Q Q Q E E Q Q Q Q Q Q E L L L K E Q L E   32 T T T P T T TT T T T G P Q E V S Q P R   33 F F F F F F F F F F F F F F F F F F F F  34 V V V T V V V V V V V T D I I F I I N D   35 Y Y Y Y Y Y Y Y Y Y YW Y Y Y Y Y Y Y W   36 G G G G G G G G G G G S S G G G G G S S   37 G GG G G G G G G G G G G G G G G G G G   38 C T A C C C C C C C C C C C C CC C C C   39 R R R Q R R R R R R R G G G G G K R G G   40 A A A G A A AA A A A G G G G G G G G G   41 K K K N K K K K K K K N N N N N N N N N  42 R R R N S R R R R R R S A A A A K Q A S   43 N N N N N N N N N N NN N N N N N N N N   44 N N N N N N N N N N N R R R R N N R R R   45 F FF F F F F F F F F F F F F F F F F F   46 K K K E K K K K K K K K K K K EK D K K   47 S S S T S S S S S S S T T T T T T T T T   48 A A A T A A AA A A A I I I I R K T I I   49 E E E E E E E E E E E E E D D D A Q E E  50 D D D M D D D D D D D E E E E E E Q E E   51 C C C C C C C C C C CC C C C C C C C C   52 M M M L M M M M M M E R R R H R V Q R R   53 R RR R R R R R R R R R R R R E R G R R   54 T T T I T T T T T T T T T T T TA V T T   55 C C C C C C C C C C C C C C C C C C C C   56 G G G E G G GG G G G I V V V G R V V I   57 G G G P G G G G G G G R G G G G P — G G  58 A A A P A A A A A A A K — — — K P — — —   59 — — — Q — — — — — — —— — — — — E — — —   60 — — — Q — — — — — — — — — — — — R — — —   61 — —— T — — — — — — — — — — — — P — — —   62 — — — D — — — — — — — — — — — —— — —   63 — — — K — — — — — — — — — — — — — — —   64 — — — S — — — — —— — — — — — — — — — BPTI Homologues (21-40) R # 21 22 23 24 25 26 27 2829 30 31 32 33 34 35 36 37 38 39 40  −5 — — — — — — — — — — — — D — — —— — — —  −4 — — — — — — — — — — — — E — — — — — — —  −3 — — — — — — — —— — — T P — — — — — — —  −2 — L Z R K — — — R R — E T — — — — — — —  −1— Q D D N — — — Q K — R T — — — Z — — —    1 R H H R R I K T R R R G D KT R R R R R    2 P R P P P N E V H H P F L A V P P P P P    3 Y T K K TG D A R P D L P D E D D D D D    4 A F F F F D S A D D F D I S A F F F FF    5 C C C C C C C C C C C C C C C C C C C C    6 E K Y Y N E Q N D DL T E Q N L L L L L    7 L L L L L L L L K K E S Q L L E E E E E    8 IP P P L P G P P P P P A D P P P P P P    9 V A A A P K Y V P P P P FG YI P P P P P   10 A E D D E V S I D D Y V D S V Y Y Y Y Y   11 A P P P TV A R K T T T A Q Q T T T T T   12 G G G G G G G G G K G G G G G G G G GG   13 P P R R R P P P N I P P L P P P P P P P   14 C C C C C C C C C CC C C C C C C C C C   15 M K K L N R M R — — K R F L R R K K K K   16 FA A A A A G A G Q A A G G A A A A A A   17 F S H Y L R M F P T K G Y L FR R R R K   18 I I I M I F T I V V M F M F I I M I M M   19 S P P P P PS Q R R I K K K Q I I I I I   20 A A R R A R R L A A R R L R L R R R R R  21 F F F F F Y Y W F F Y Y Y Y W Y Y Y Y Y   22 Y Y Y Y Y Y F A Y Y FN S F A F F F F F   23 Y Y Y Y Y Y Y F Y Y Y Y Y Y F Y Y Y Y Y   24 S ND N N N N D D K N N N N D N N N N N   25 K W S P S S G A T P A T Q G A AA A A A   26 G A A A H S T V R S K R E T V K K K K K   27 A A S S L S SK L A A T T S K A A A A A   28 N K N N H K M G K K G K K M G G G G G G  29 K K K K K R A K T R F Q N A K L L L L F   30 C C C C C C C C C C CC C C C C C C C C   31 Y Q N E Q E E V K V E E E E V Q Q Q Q E   32 P LK K K K T L A Q T P E T R T P P P T   33 F F F F F F F F F F F F F F F FF F F F   34 T H I I N I Q P Q R V K I L S V V V V V   35 Y Y Y Y Y Y YY Y Y Y Y Y Y Y Y Y Y Y Y   36 S G G G G G G G R G G G G G G G G G G G  37 G G G G G G G G G G G G G G G G G G G G   38 C C C C C C C C C C CC C C C C C C C C   39 R K P R G G M Q D D K K Q M K R R R R K   40 G GG G G G G G G G A G G G G A A A A A   41 N N N N N N N N D D K N N N N KK K K K   42 A A A A A A G G H H S G D L G R S R R S   43 N N N N N N NN G G N N N N N N N N N N   44 R R N N N N N K N N N R R N K N N N N N  45 F F F F F F F F F F F Y F F F F F F F F   46 K S K K K H V Y K K RK S L Y K K K K R   47 T T T T T T T S T S S S T S S S S S S S   48 I IW W I L E E E D A E L Q Q A A S A A   49 E E D D D E K K T H E Q A K K EE E E E   50 E K E E E E E E L L D D E E E D D D D D   51 C C C C C C CC C C C C C C C C C C C C   52 R R R R Q E L R R R M L E L K E M M M M  53 R H Q H R K Q E C C R D Q Q E R R R R R   54 T A T T T V T Y E E TA K T Y T T T T T   55 C C C C C C C C C C C C C C C C C C C C   56 V VG V A G R G L E G S I R G G G G G G   57 V G A A A V — V V L G G N — I GG G G G   58 — — S S K R — P Y Y A F — — P A A A A A   59 — — A G Y S —G P R — — — — G — — — — —   60 — — — I G — — D — — — — — — E — — — — —  61 — — — — — — — E — — — — — — A — — — — — Legand To Table 13  1 BPTI 2 Engineered BPTI From MARK87  3 Engineered BPTI From MARK87  4 BovineColostrum (DUFT85)  5 Bovine Serum (DUFT85)  6 Semisynthetic BPTI,TSCH87  7 Semisynthetic BPTI, TSCH87  8 Semisynthetic BPTI, TSCH87  9Semisynthetic BPTI, TSCH87 10 Semisynthetic BPTI, TSCH87 11 EngineeredBPTI, AUER87 12 Dendroaspis polylepis polylepis (Black mamba) venom I(DUFT85) 13 Dendroaspis polylepis polylepis (Black Mamba) venom K(DUFT85) 14 Hemachatus hemachates (Ringhals Cobra) HHV II (DUFT85) 15Naja nivea (Cape cobra) NNV II (DUFT85) 16 Vipera russelli (Russel'sViper) RVV II (TAKA74) 17 Red sea turtle egg white (DUFT85) 18 Snailmucus (Helix pomania) (WAGN78) 19 Dendroaspis angusticeps (Eastern greenmamba) C13 S1 C3 toxin (DUFT85) 20 Dendroaspis angusticeps (EasternGreen Mamba) C13 S2 C3 toxin (DUFT85) 21 Dendroaspis polylepis polylepes(Black mamba) B toxin (DUFT85) 22 Dendroaspis polylepis polylepes (BlackMamba) E toxin (DUFT85) 23 Vipera ammodytes TI toxin (DUFT85) 24 Viperaammodytes CTI toxin (DUFT85) 25 Bungarus fasciatus VIII B toxin (DUFT85)26 Anemonia sulcata (sea anemone) 5 II (DUFT85) 27 Homo sapiens HI-14“inactive” domain (DUFT85) 28 Homo sapiens HI-8 “active” domain (DUFT85)29 beta bungarotoxin B1 (DUFT85) 30 beta bungarotoxin B2 (DUFT85) 31Bovine spleen TI II (FIOR85) 32 Tachypleus tridentatus (Horseshoe crab)hemocyte inhibitor (NAKA87) 33 Bombyx mori (silkworm) SCI-III (SASA84)34 Bos taurus (inactive) BI-14 35 Bos taurus (active) BI-8 36:Engineered BPTI (KR15, ME52): Auerswald '88, Biol Chem Hoppe-Seyler, 369Supplement, pp27-35. 37: Isoaprotinin G-1: Siekmann, Wenzel, Schroder,and Tschesche '88, Biol Chem Hoppe-Seyler, 369: 157-163. 38:Isoaprotinin 2: Siekmann, Wenzel, Schroder, and Tschesche '88, Biol ChemHoppe-Seyler, 369: 157-163. 39: Isoaprotinin G-2: Siekmann, Wenzel,Schroder, and Tschesche '88, Biol Chem Hoppe-Seyler, 369: 157-163. 40:Isoaprotinin 1: Siekmann, Wenzel, Schroder, and Tschesche '88, Biol ChemHoppe-Seyler, 369: 157-163.

[0237] TABLE 30 Tables: IIIsp::bpti::mautreIII (initial fragment) fusiongene. The DNA sequence has SEQ ID NO. 001; Amino-acid sequence has SEQID NO. 002. The DNA is linear and is shown on the lines that do notbegin with “!”. The DNA encoding mature III is identical to the DNAfound in M13mp18. The amino-acid sequence is processed in vivo anddisulfide bonds form. !    m   k   k   l   l   f   a   I   p   l SEQ IDNO. 002 !    1   2   3   4   5   6   7   8   9  10  5′-gtg aaa aaa ttatta ttc gca att cct tta SEQ ID NO. 001 !  |<---- gene III signalpeptide-------- ! !                                    - cleavage site!                                    |!                                    ↓!       v   v   p   f   y   s   G   A!      11  12  13  14  15  16  17  18        gtt gtt cct ttc tat tct GGcGcc !      ----------------------------->| !!                 | R | P | D | F | C | L | E |!                 | 19| 20| 21| 22| 23| 24| 25|                  |CGT|CCG|GAT|TTC|TGT|CTC|GAG|- ! M13/BPTIJnct   ↑  |AccIII|         |XhoI  |(& AvaI)! !! | P | P | Y | T | G | P | C | K | A | R | ! | 26| 27| 28| 29| 3031| 32| 33| 34| 35|   |CCA|CCA|TAC|ACT|GGG|CCC|TGC|AAA|GCG|CGC|-!     |PflMI      |     ||      |BssHII | !                 | ApaI  |!                 | DraII  | = PssI !! | I | I | R | Y | F | Y | N | A | K | A |! | 36| 37| 38| 39| 40| 41| 42| 43| 44| 45|  |ATC|ATC|CGC|TAT|TTC|TAC|AAT|GCT|AAA|GC|- !! | G | L | C | Q | T | F | V | Y | G | G | ! | 46| 47| 48| 49| 5051| 52| 53| 54| 55|  A|GGC|CTG|TGC|CAG|ACC|TTT|GTA|TAC|GGT|GGT|-!| StuI |                 |XcaI  |( & AccI) !! | C | R | A | K | R | N | N | F | K |! | 56| 57| 58| 59| 60| 61| 62| 63| 64|  |TGC|CGT|GCT|AAG|CGT|AAC|AAC|TTT|AAA|- !         | EspI   | !! | S | A | E | D | C | M | R | T | C | G | ! | 65| 66| 6768| 69| 70| 71| 72| 73| 74|   |TCG|GCC|GAA|GAT|TGC|ATG|CGT|ACC|TGC|GGT|-!   |XmaIII|        | SphI | ! !         BPTI/M13 boundary! | G | A | A   E   (Residue numbers of mature III have had! | 75| 76|119 120  118 added to the usual residue numbers.)  |GGC|GCC|gct gaa ! | NarI  | (& KasI) ! ! 121 122 123 124 125 126 127128 129 130 131 132 133 134!  T   V   E   S   C   L   A   K   P   H   T   E   N   S ...   act gttgaa agt tgt tta gca aaa ccc cat aca gaa aat tca... ! ! The remainder ofthe gene is identical to the corresponding part of iii in M13 mp18.

[0238] TABLE 35 IIIsp::itiDl::matureIII fusion gene. DNA has SEQ ID NO.003; amino-acid sequence has SEQ ID NO. 004. The DNA is a linear segmentand the amino-acid sequence is a protein that is processed in vivo andwhich contains disulfides. m   k   k   l   l   f   a   I   p   l   v   v   p   f   y SEQ ID NO.004 −18 −17 −16 −15 −14 −13 −12 −11 −10  −9  −8  −7  −6  −5  −4 5′-gtgaaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat |<---- gene IIIsignal peptide --------------------------- SEQ ID NO. 003             -cleavage site            | s   G   A   K   E   D   S   C   Q   L   G   Y   S   A   G−3  −2  −1  1   2   3   4   5   6   7   8   9   10  11  12 tct GGc Gccaaa gaa gaC tcT tGC CAG CTG GGC tac tCG GCC Ggt --------->|                 |  BglI      |      |EagI |     | KasI | 13  14  15  16  17  18  19  20  21  22  23  24  25  26 P   C   M   G   M   T   S   R   Y   F   Y   N   G   T ccc tgc atg ggaatg acc agc agg tat ttc tat aat ggt aca 27  28  29  30  31  32  33  34  35  36  37  38  39  40  41 S   M   A   C   E   T   F   Q   Y   G   G   C   M   G   N tCC ATG Gcctgt gag act ttc cag tac ggc ggc tgc atg ggc aac  | NcoI |  | StyI | 42  43  44  45  46  47  48  49  50  51  52  53  54  55  56 G   N   N   F   V   T   E   K   E   C   L   Q   T   C   R ggt aac aacttc gtc aca gaa aag gag tgt CTG CAG acc tgc cga                                       | PstI  |  57  58    101 102 119120  T   V    g   a   A   E act gtg ggc gcc gct gaa          | BbeI  |   (Residue numbers of mature          | NarI  |   III have had 118 added to          | KasI  |   the usual residue numbers.) 121 122 123 124 125126 127 128 129 130 131 132 133 134 135 T   V   E   S   C   L   A   K   P   H   T   E   N   S   F.. act gtt gaaagt tgt tta gca aaa ccc cat aca gaa aat tca ttt.. The remainder of thegene is identical to the corresponding part of gene iii in phageM13mp18.

[0239] TABLE 55 Affinity Classes of ITI-D1-derived hNE inhibitorsFraction of pH Affinity Estimated Input Elution Class K_(D) boundMaximum Protein WEAK K_(D) > 10 nM <0.005% >6.0 ITI-D1 MODERATE  1 to 10nM 0.01% to 5.5 to 5.0 BITI 0.03% ITI-D1E7 STRONG 10 to 1000 0.03% to5.0 to 4.5 BITI-E7 pM 0.06% BITI-E7-1222 AMINO1 AMINO2 MUTP1 VERY <10 pM >0.1% ≦4.0 BITI-E7-141 STRONG MUTT26A MUTQE MUT1619

[0240] TABLE 65 Definition of Class A, B and C mutations inPCT/US92/01501. Classes: A No major effect expected if molecular chargestays in range −1 to +1. B Major effects not expected, but are morelikely than in “A”. C Residue in the binding interface; any change mustbe tested. X No substitution allowed. Res. Id. EpiNE1 SubstitutionsClass  1 R any A  2 P any A  3 D any A  4 F Y, W, L B  5 C C X  6 Lnon-proline A  7 E L, S, T, D, N, K, R A  8 P any A  9 P any A 10 Ynon-proline prefr'd B 11 T any C 12 G must be G X 13 P any C 14 C Cstrongly preferred, any non-proline C 15 I V, A C 16 A C 17 F L, I, M,Y, W, H, V C 18 F Y, W, H C 19 P any C 20 R non-proline prefr'd C 21 Y F& Y most prefr'd; W, I, L prefr'd; M, V C allowed 22 F Y & F mostprefr'd; non-proline prefr'd Y, F B 23 Y Y & F strongly prefr'd F, Y B24 N non-proline prefr'd A 25 A any A 26 K any A 27 A any A 28 Gnon-proline prefr'd A 29 L non-proline prefr'd A 30 C must be C X 31 Qnon-proline prefr'd B 32 T non-proline prefr'd B 33 F F very stronglyprefr'd; Y possible X 34 V any C 35 Y Y most prefr'd; W prefr'd; Fallowed B 36 G G strongly prefr'd; S, A prefr'd; C 37 G must be G solong as 38 is C X 38 C C strongly prefr'd X 39 M any C 40 G A, S, N, D,T, P C 41 N K, Q, S, D, R, T, A, E C 42 G any C 43 N must be N X 44 N S,K, R, T, Q, D, E B 45 F Y B 46 K any non-proline B 47 ST, N, A, G B 48 Aany B 49 E any A 50 D any A 51 C must be C X 52 M any A 53 R any A 54 Tany A 55 C must be C X 56 G any A 57 G any A 58 A any A

[0241] TABLE 100 Sequences of Kunitz domains Sequence Seq          111111111122222222223333333333444  4444444555555555 ParentalId Name 123456789a012345678901234567890123456789012ab345678 domain No.Consensus RPDFCLLPA-ETGPGRAMTPRFYYNAKSGKCEPFTYGGCGGNA--NNFKTEEECRRTCGGA005 Kunitz  1         3      5              7      9 Domain      2       4           6           8           10          BPTIRPDFCLEPP-YTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKR--NNFKSAEDCMRTCGGA BPTI 006(Genebank P00974) EPI-rpdfclepp-ytgpcIaFFPryfynakaglcqtfvyggcCMGNG--nnfksaedcmrtcgga BPTI 007HNE-1 = EpiNE1 EPI-HNE-2EAEArpdfclepp-ytgpcIaFFPryfynakaglcqtfvyggCMGNG--nnfksaedcmrtcgga BPTI008 EpiNE7    rpdfclepp-ytgpcVaMFPryfynakaglcqtfvyggcMGNG--nnfksaedcmrtcgga BPTI009 EpiNE3    rpdfclepp-ytgpcVGFFSryfynakaglcqtfvyggcMGNG--nnfksaedcmrtcgga BPTI010 EpiNE6    rpdfclepp-ytgpcVGFFQryfynakaglcqtfvyggcMGNG--nnfksaedcmrtcgga BPTI011 EpiNE4    rpdfclepp-ytgpcVaIFPryfynakaglcqtfvyggcMGNG--nnfksaedcmrtcgga BPTI012 EpiNE8    rpdfclepp-ytgpcVaFFKrstynakaglcqtfvyggcMGNG--nnfksaedcmrtcgga BPTI013 EpiNE5    rpdfclepp-ytgpclaFFQryfynakaglcqtfVyggcMGNG--nnfksaedcmrtcgga BPTI014 EpiNE2    rpdfclepp-ytgpcIaLFKryfynakaglcqtfvyggcMGNG--nnfksaedcmrtcgga BPTI015 ITI-D1 KEDSCQLGY-SAGPCMGMTSRYFYNGTSMACETFQYGGCMGNG--NNFVTEKDCLQTCRTVITI-D1 016 (Genebank P02760) BITI-RPdFcqlgy-sagpcVAmFPryfyngtsmacQtfVyggcmgng--nnfvtekdclqtcrga ITI-D1 017E7-141 MUTT26ARPdFcqlgy-sagpcVAmEPryfyngAsmacQtfVyggcmgng--nnfvtekdclqtcrga ITI-D1 018MUTQE RPdFcqlgy-sagpcVAmEPryfyngtsmacetfVyggcmgng--nnfvtekdclqtcrgaITI-D1 019 MUT1619RPdFcqlgy-sagpcVgmFsryfyngtsmacQtfVyggcmgng--nnfvtekdclqtcrga ITI-D1 020ITI-D1E7 kedscqlgy-sagpcVAmFPryfyngtsmacetfqyggcmgng--nnfvtekdclqtcrgaITI-D1 021 AMINO1kedFcqlgy-sagpcVAmFPryfyngtsmacetfqyggcmgng--nnfvtekdclqtcrga ITI-D1 022AMINO2 kpdscqlgy-sagpcVAmFPryfyngtsmacetfqyggcmgng--nnfvtekdclqtcrgaITI-D1 023 MUTP1RPdFcqlgy-sagpclgmFsryfyngtsmacetfqyggcmgng--nnfvtekdclqtcrga ITI-D1 024ITI-D2 TVAACNLPI-VRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNG--NKFSEKECGVP ITI-D2025 (Genebank P02760) EPI-HNE-3aacnlpi-vrgpcIafFPRwafdavkgkcvlfpyggcqgng--nkfysekecreycgvp ITI-D2 026EPI-HNE-4 Eacnlpi-vrgpcIafFPRwafdavkgkcvlfpyggcqgng--nkfysekecreycgvpITI-D2 027 App-IVREVCSEQA-ETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNR--NNFDTEEYCMAVCGSA 028 (NCBI105306) DPI.1.1vrevcseqa-YtgpcIaFFPrYyfdvtegkcQTfvyggcMgnG--nnfdteeycmavcgsa APP-I 029DPI.1.2 vrevcsega-etgpcIamFsrwyfdvtegkcapfVyggcggnr--nnfdteeycmavcgsaAAP-I 030 DPI.1.3vrevcseqa-etgpcIaFFsrwyfdvtegkcaTfVyggcMgnr--nnfdteeycmavcgsa AAP-I 031TFPI2-D1 NAEICLLPL-DYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNA--NNFYTWEACDDACWRI032 (SPRE94) DPI.2.1naeicllpl-YTgpcIaFFPryyydrytqscQTfVyggcMgna--nnfytweacddacwri TFPI2-D1033 DPI.2.2naeicllpl-dygpcIalFlryyydrytgscrqfVyggcegna--nnfytweacddacwri TFPI2-D1034 DPI.2.3naeicllpl-dTgpcIaFFlryyydrytqscQTfvyggcMgna--nnfytweacddacwri TFPI2-D1035 TFPI2-D2VPKVCRLQVSVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNRIENRFPDEATCMGFCAPK 036(SPRE94) DPI.3.1vpkvcrlqv-vRGPcIAFFPRWffnlssmtcvLfPYggcQGnG--nrfpdeatcmgfcapk 037DPI.3.2 vpkvcrlqvsvddqcIgsFekyffnlAsmtceTfVsggchrnrienrfpdeatcmgfcapkTFPI2-D1 038 DPI.3.3vpkvcrlqv-vAGPcIgFFKRyffAlssmtceTfvsggchrnr--nrfpdeatcmgfcapk TFPI2-D1039 TFPI2-D3ipsfcyspk-deglcsanvtryyfnpryrtcdaftytgcggnd--nnfvsredckracaka 040(SPRE94) DPI.4.1ipsfcyspk-SAgPcVaMFPryyfnpryrtcETfvyGgcMgnG--nnfvsredckracaka TFPI2-D3041 DPI.4.2ipsfcyspk-deglcIavFtryyfnpryrtcdaftytgcggnd--nnfvsredckracaka TFPI2-D3042 DPI.4.3ipsfcyspk-dTgPcIaFFtryyfnpryrtcdTfvyGgcggnd--nnfvsredckracaka TFPI2-D3043 LACI-D1mhsfcafka-ddgpckaimkrfffniftrqceefiyggcegnq--nrfesleeckkmctrd 044(Genebank P10646) DPI.5.1mhsfcafka-SAgpcVaMFPryffniftrqceTfVyggcMgnG--nrfesleeckkmctrd LACI-D1045 DPI.5.2mhsfcafka-ddgpcIaiFkrfffniftrqceefiyggcegnq--nrfesleeckkmctrd LACI-D1046 DPI.5.3mhsfcafka-YTgpcIavvkrfffniftrqceTfiyggcegnq--nrfesleeckkmctrd LACI-D1047 LACI-D2KPDFCFLEE-DPGICRGYITRYFYNNQTKQCERFKYGGCLGNM--NNFETLEECKNICEDG 048(Genebank P10646) DPI.6.1kpdfcflee-SAgPcVAMFPryfynnqtkqceTfVyggcMqnG--nnfetleecknicedg LACI-D2049 DPI.6.2kpdfcflee-dpgicVgyFtryfynnqtkqcerfkyggclgnm--nnfetleecknicedg LACI-D2050 DPI.6.3kpdfcflee-dpgicvgFFtryfynnqtkqcerfVyggclgnm--nnfetleecknicedg LACI-D2051 DPI.6.4kpdfcflee-dpgicvgFFtryfynAqtkqcerfVyggclgnm--nnfetleecknicedg LACI-D2052 DPI.6.5kpdfcflee-dpgPcVgFFQryfynAqtkqcerfVyggcQgnm--nnfetleecknicedg LACI-D2053 DPI.6.6kpdfcflee-dpgPcVgFFtryfynnqtkqcerfVyggcQgnm--nnfetleecknicedg LACI-D2054 DPI.6.7kpdfcflee-dpgPcIgFFPryfynnqtkqcerfvyggcQgnm--nnfetleecknicedg LACI-D2055 LACI-D3GPSWCLTPA-DRGLCRANENRFYYNSVIGKCRPFKYSGCGCNE--NNFTSKQECLRACKKG 056(Genebank P10646) DPI.7.1gpswcltpa-VrgPcIaFFPrWyynsvigkcVLfpyGgcQgnG--nnftskqeclrackkg LACI-D3057 DPI.7.2gpswcltpa-drglcVanFnrfyynsvigkcrpfkysgcggne--nnftskqeclrackkg LACI-D3058 DPI.7.3gpswcltpa-drglcVaFFnrfyynsvigkcrpfkysgcggne--nnftskqeclrackkg LACI-D3059 DPI.7.4gpswcltpa-VrgPcVaFFnrfyynsvigkcrpfkyGgcggne--nnftskqeclrackkg LACI-D3060 DPI.7.5gpswcltpa-drgPcIaFFPrWyynsvigkcQTfVyGgcggne--nnftskqeclrackkg LACI-D3061 A3 ETDICKLPK-DEGTCRDFILKWYYDPNTKSCARFWYGGCGGNE--NKFGSQKECEKVCAPV 062collagen (W093/ 14119) DPI.8.1etdicklpk-VRgPcIAfFPRwyydpntkscVLfpyggcQgnG--nkfgsqkecekvcapv A3 063DPI.8.2 etdicklpk-degtcIAfFlkwyydpntkscarfVyggcggne--nkfgsqkecekvcapv A3064 collagen DPI.8.3etdicklpk-degPcIAfFlRwyydpntkscarfVyggcggne--nkfgsqkecekvcapv A3 065 HKIB9 LPNVCAFPM-EKGPCQTYMTRWFFNFETGECELFAYGGCGGNS--NNFLRKEKCEKFCKFT 066Domain (NORR93) DPI.9.1lpnvcafpm-VRgpcIAFFPrwffnfetgecVlfVyggcQgnG--nnflrkekcekfckft HKI B9 067DPI.9.2 lpnvcafpm-ekgpcIAyFtrwffnfetgecelfayggcggns--nnflrkekcekfckftHKI B9 068 DPI.9.3lpnvcafpm-ekgpcIAyFPrwffnfetgecVlfVyggcggns--nnflrkekcekfckft HKI B9 069

[0242] TABLE 111 Restriction sites in plasmid pHIL-D2 pHIL-D2, 93-01-02Ngene = 8157 Non-cutters AflII ApaI AscI AvaI AvrII BamHI BglII Bsp120IBsrGI BssHII BstEII FseI MluI NruI PacI PmlI RsrII SacII SexAI SfiI SgfISnaBI SpeI Sse8387I XhoI XmaI (PaeR7I) (SmaI) Cutters AatII GACGTc 15498 AflIII Acrygt 1 7746 AgeI Accggt 1 1009 BlpI GCtnagc 1 597 BspEI(BspMII,AccIII) Tccgga 1 3551 BspMI gcaggt 1 4140 Bst1107I GTAtac 1 7975BstBI (AsuII) TTcgaa 2 945 4780 Bsu36I CCtnagg 1 1796 Ec1136I GAGctc 1216 EcoRI Caattc 1 956 EspI (Bpu1102I) GCtnagc 1 597 HpaI GTTaac 1 1845NcoI Ccatgg 1 3339 NdeI CAtatg 1 7924 NsiI (Ppu10I) ATGCAt 1 684 PflMICCANNNNntgg 1 196 PmeI GTTTaaac 1 420 PstI CTGCAg 1 6175 PvuI CGATcg 16049 SapI gaagagc 1 7863 SacI GAGCTc 1 216 SalI Gtcgac 1 2885 ScaIAGTact 1 5938 SphI GCATGc 1 4436 Stul AGGcct 1 2968 SwaI ATTTaaat 1 6532TthlllI GACNnngtc 1 7999 XbaI Tctaga 1 1741 XcmI CCANNNNNnnnntgg 1 711Aox1 5′    1 to about 950 Aox1 3   950 to about 1250 His4 1700 to about4200 Aox1 3′ 4500 to 5400 bla 5600 to 6400 f1 ori 6500 to 6900

[0243] TABLES 207-208 (merged) SEQUENCES OF THE EpiNE CLONES TN THE P1REGION SEQUENCE CLONE 1 1 1 1 1 1 1 2 2 IDENTIFIERS 3 4 5 6 7 8 9 0 1BPTI (comp. P C K A R I I R Y (BPTI) only) (SEQ ID NO:6) P C V A M F Q RY EpiNEα 3, 9, 16, 17, P C V G F F S R Y EpiNE3 18, 19 (SEQ ID NO:10) 6P C V G F F Q R Y EpiNE6 (SEQ ID NO:11) 7, 13, 14, 15, P C V A M F P R YEpiNE7 20 (SEQ ID NO:9) 4 P C V A I F P R Y EpiNE4 (SEQ ID NO:12) 8 P CV A I F K R S EpiNE8 (SEQ ID NO:13) 1, 10, 11, 12 P C I A F F P R YEpiNEl (SEQ ID NO:7) 5 P C I A F F Q R Y EpiNE5 (SEQ ID NO:14) 2 P C I AL F K R Y EpiNE2 (SEQ IDN O:15)

[0244] TABLE 212 Fractionation of EpiNE-7 and MA-ITI-D1 phage on hNEbeads EpiNE-7 MA-ITI-D1 pfu pfu/INPUT pfu pfu/INPUT INPUT 3.3 · 10⁹ 1.00 3.4 · 10¹¹ 1.00 Final 3.8 · 10⁵ 1.2 · 10⁻⁴ 1.8 · 10⁶ 5.3 · 10⁻⁶TBS-TWEEN Wash pH 7.0 6.2 · 10⁵ 1.8 · 10⁻⁴ 1.6 · 10⁶ 4.7 · 10⁻⁶ 6.0 1.4· 10⁶ 4.1 · 10⁻⁴ 1.0 · 10⁶ 2.9 · 10⁻⁶ 5.5 9.4 · 10⁵ 2.8 · 10⁻⁴ 1.6 · 10⁶4.7 · 10⁻⁶ 5.0 9.5 · 10⁵ 2.9 · 10⁻⁴ 3.1 · 10⁵ 9.1 · 10⁻⁷ 4.5 1.2 · 10⁶3.5 · 10⁻⁴ 1.2 · 10⁵ 3.5 · 10⁻⁷ 4.0 1.6 · 10⁶ 4.8 · 10⁻⁴ 7.2 · 10⁴ 2.1 ·10⁻⁷ 3.5 9.5 · 10⁵ 2.9 · 10⁻⁴ 4.9 · 10⁴ 1.4 · 10⁻⁷ 3.0 6.6 · 10⁵ 2.0 ·10⁻⁴ 2.9 · 10⁴ 8.5 · 10⁻⁸ 2.5 1.6 · 10⁵ 4.8 · 10⁻⁵ 1.4 · 10⁴ 4.1 · 10⁻⁸2.0 3.0 · 10⁵ 9.1 · 10⁻⁵ 1.7 · 10⁴ 5.0 · 10⁻⁸ SUM 6.4 · 10⁶   3 · 10⁻³5.7 · 10⁶   2 · 10⁻⁵

[0245] TABLE 214 Abbreviated fractionation of display phage on hNE beadsDisplay phage MA-ITI- EpiNE-7 MA-ITI-D1 2 MA-ITI-D1E7 1 D1E7 2 INPUT1.00 1.00 1.00 1.00 (pfu) (1.8 × 10⁹) (1.2 × 10¹⁰ (3.3 × 10⁹) (1.1 ×10⁹) Wash  6 · 10⁻⁵  1 · 10⁻⁵  2 · 10⁻⁵  2 · 10⁻⁵ pH 7.0  3 · 10⁻⁴  1 ·10⁻⁵  2 · 10⁻⁵  4 · 10⁻⁵ pH 3.5  3 · 10⁻³  3 · 10⁻⁶  8 · 10⁻⁵  8 · 10⁻⁵pH 2.0  1 · 10⁻³  1 · 10⁻⁶  6 · 10⁻⁶  2 · 10⁻⁵ SUM  4.3 · 10⁻³  1.4 ·10⁻⁵  1.1 · 10⁻⁴  1.4 · 10⁻⁴

[0246] TABLE 215 Fractionation of EpiNE-7 and MA-ITI-D1E7 phage on hNEbeads EpiNE-7 MA-ITI-D1E7 Fraction of Fraction of Total pfu Input Totalpfu Input INPUT 1.8 · 10⁹ 1.00 3.0 · 10⁹ 1.00 pH 7.0 5.2 · 10⁵ 2.9 ·10⁻⁴ 6.4 · 10⁴ 2.1 · 10⁻⁵ pH 6.0 6.4 · 10⁵ 3.6 · 10⁻⁴ 4.5 · 10⁴ 1.5 ·10⁻⁵ pH 5.5 7.8 · 10⁵ 4.3 · 10⁻⁴ 5.0 · 10⁴ 1.7 · 10⁻⁵ pH 5.0 8.4 · 10⁵4.7 · 10⁻⁴ 5.2 · 10⁴ 1.7 · 10⁻⁵ pH 4.5 1.1 · 10⁶ 6.1 · 10⁻⁴ 4.4 · 10⁴1.5 · 10⁻⁵ pH 4.0 1.7 · 10⁶ 9.4 · 10⁻⁴ 2.6 · 10⁴ 8.7 · 10⁻⁶ pH 3.5 1.1 ·10⁶ 6.1 · 10⁻⁴ 1.3 · 10⁴ 4.3 · 10⁻⁶ pH 3.0 3.8 · 10⁵ 2.1 · 10⁻⁴ 5.6 ·10³ 1.9 · 10⁻⁶ pH 2.5 2.8 · 10⁵ 1.6 · 10⁻⁴ 4.9 · 10³ 1.6 · 10⁻⁶ pH 2.02.9 · 10⁵ 1.6 · 10⁻⁴ 2.2 · 10³ 7.3 · 10⁻⁷ SUM 7.6 · 10⁶ 4.1 · 10⁻³ 3.1 ·10⁵ 1.1 · 10⁻⁴

[0247] TABLE 216 Fractionation of MA-EpiNE-7, MA-BITI and MA-BITI-E7 onhNE beads MA-BITI MA-BITI-E7 MA-EpiNE7 pfu pfu/Input pfu pfu/Input pfupfu/Input INPUT pH 2.0 10¹⁰ 1.00 6.0 10⁹ 1.00 1.5 10⁹ 1.00 7.0 2.4 10⁵1.2 10⁻⁵ 2.8 10⁵ 4.7 10⁻⁵ 2.9 10⁵ 1.9 10⁻⁴ 6.0 2.5 10⁵ 1.2 10⁻⁵ 2.8 10⁵4.7 10⁻⁵ 3.7 10⁵ 2.5 10⁻⁴ 5.0 9.6 10⁴ 4.8 10⁻⁶ 3.7 10⁵ 6.2 10⁻⁵ 4.9 10⁵3.3 10⁻⁴ 4.5 4.4 10⁴ 2.2 10⁻⁶ 3.8 10⁵ 6.3 10⁻⁵ 6.0 10⁵ 4.0 10⁻⁴ 4.0 3.110⁴ 1.6 10⁻⁶ 2.4 10⁵ 4.0 10⁻⁵ 6.4 10⁵ 4.3 10⁻⁴ 3.5 8.6 10⁴ 4.3 10⁻⁶ 9.010⁴ 1.5 10⁻⁵ 5.0 10⁵ 3.3 10⁻⁴ 3.0 2.2 10⁴ 1.1 10⁻⁶ 8.9 10⁴ 1.5 10⁻⁵ 1.910⁵ 1.3 10⁻⁴ 2.5 2.2 10⁴ 1.1 10⁻⁶ 2.3 10⁴ 3.8 10⁻⁶ 7.7 10⁴ 5.1 10⁻⁵ 2.07.7 10³ 3.8 10⁻⁷ 8.7 10³ 1.4 10⁻⁶ 9.7 10⁴ 6.5 10⁻⁵ SUM 8.0 10⁵ 3.9 10⁻⁵1.8 10⁶ 2.9 10⁻⁴ 3.3 10⁶ 2.2 10⁻³

[0248] TABLE 217 Fractionation of MA-BITI-E7 and MA-BITI-E7-1222 on hNEbeads MA-BITI-E7 MA-BITI-E7-1222 pfu pfu/INPUT pfu pfu/INPUT INPUT 1.3 ·10⁹ 1.00 1.2 · 10⁹ 1.00 pH 7.0 4.7 · 10⁴ 3.6 · 10⁻⁵ 4.0 · 10⁴ 3.3 · 10⁻⁵6.0 5.3 · 10⁴ 4.1 · 10⁻⁵ 5.5 · 10⁴ 4.6 · 10⁻⁵ 5.5 7.1 · 10⁴ 5.5 · 10⁻⁵5.4 · 10⁴ 4.5 · 10⁻⁵ 5.0 9.0 · 10⁴ 6.9 · 10⁻⁵ 6.7 · 10⁴ 5.6 · 10⁻⁵ 4.56.2 · 10⁴ 4.8 · 10⁻⁵ 6.7 · 10⁴ 5.6 · 10⁻⁵ 4.0 3.4 · 10⁴ 2.6 · 10⁻⁵ 2.7 ·10⁴ 2.2 · 10⁻⁵ 3.5 1.8 · 10⁴ 1.4 · 10⁻⁵ 2.3 · 10⁴ 1.9 · 10⁻⁵ 3.0 2.5 ·10³ 1.9 · 10⁻⁶ 6.3 · 10³ 5.2 · 10⁻⁶ 2.5 <1.3 · 10³  <1.0 · 10⁻⁶  <1.3 ·10³  <1.0 · 10⁻⁶  2.0 1.3 · 10³ 1.0 · 10⁻⁶ 1.3 · 10³ 1.0 · 10⁻⁶ SUM 3.8· 10⁵ 2.9 · 10⁻⁴ 3.4 · 10⁵ 2.8 · 10⁻⁴

[0249] TABLE 218 Fractionation of MA-EpiNE7 and MA-BITI-E7-141 on hNEbeads MA-EpiNE7 MA-BITI-E7-141 pfu pfu/INPUT pfu pfu/INPUT INPUT 6.1 ·10⁸ 1.00 2.0 · 10⁹ 1.00 pH 7.0 5.3 · 10⁴ 8.7 · 10⁻⁵ 4.5 · 10⁵ 2.2 · 10⁻⁴6.0 9.7 · 10⁴ 1.6 · 10⁻⁴ 4.4 · 10⁵ 2.2 · 10⁻⁴ 5.5 1.1 · 10⁵ 1.8 · 10⁻⁴4.4 · 10⁵ 2.2 · 10⁻⁴ 5.0 1.4 · 10⁵ 2.3 · 10⁻⁴ 7.2 · 10⁵ 3.6 · 10⁻⁴ 4.51.0 · 10⁵ 1.6 · 10⁻⁴ 1.3 · 10⁶ 6.5 · 10⁻⁴ 4.0 2.0 · 10⁵ 3.3 · 10⁻⁴ 1.1 ·10⁶ 5.5 · 10⁻⁴ 3.5 9.7 · 10⁴ 1.6 · 10⁻⁴ 5.9 · 10⁵ 3.0 · 10⁻⁴ 3.0 3.8 ·10⁴ 6.2 · 10⁻⁵ 2.3 · 10⁵ 1.2 · 10⁻⁴ 2.5 1.3 · 10⁴ 2.1 · 10⁻⁵ 1.2 · 10⁵6.0 · 10⁻⁵ 2.0 1.6 · 10⁴ 2.6 · 10⁻⁵ 1.0 · 10⁵ 5.0 · 10⁻⁵ SUM 8.6 · 10⁵1.4 · 10⁻³ 5.5 · 10⁶ 2.8 · 10⁻³

[0250] TABLE 219 pH Elution Analysis of hNE Binding by BITI-E7-141Varient Display Phage Fraction of Input Input recovered at pH RecoveryDisplayed PFU pH pH 3.5 × pH 2.0 × Total × protein (× 10⁹) 7.0 10⁻⁴ 10⁻⁴10⁻⁴ Relative AMINO1 0.96 0.24 2.3 0.35 2.9 0.11 (EE) AMINO2 6.1 0.572.1 0.45 3.1 0.12 (AE) BITI-E7- 1.2 0.72 4.0 0.64 5.4 0.21 1222 (EE)EpiNE7 0.72 0.44 6.4 2.2 9.0 0.35 (EE) MUTP1 3.9 1.8 9.2 1.2 12.0 0.46(AE) MUT1619 0.78 0.82 9.9 0.84 12.0 0.46 (EE) MUTQE 4.7 1.2 16. 5.322.0 0.85 (AE) MUTT26A 0.51 2.5 19.0 3.3 25.0 0.96 (EE) BITI-E7- 1.7 2.218.0 5.4 26.0 1.00 141 (AE) BITI-E7- 0.75 2.1 21. 3.2 26.0 1.00 141 (EE)

[0251] TABLE 250 Plasmid pHIL-D2 SEQ ID NO. 070 8157 base pairs. Onlyone strand is shown, but the DNA exists as double-stranded circular DNAin vivo.          1          2          3          4          51234567890 1234567890 1234567890 1234567890 1234567890 1 AgATCgCggCCgCgATCTAA CATCCAAAgA CgAAAggTTg AATgAAACCT 51 TTTTgCCATC CgACATCCACAggTCCATTC TCACACATAA gTgCCAAACg 101 CAACAggAgg ggATACACTA gCAgCAgACCgTTgCAAACg CAggACCTCC 151 ACTCCTCTTC TCCTCAACAC CCACTTTTgC CATCgAAAAACCAgCCCAgT 201 TATTgggCTT gATTggAgCT CgCTCATTCC AATTCCTTCT ATTAggCTAC251 TAACACCATg ACTTTATTAg CCTgTCTATC CTggCCCCCC TggCgAggTC 301ATgTTTgTTT ATTTCCgAAT gCAACAAgCT CCgCATTACA CCCgAACATC 351 ACTCCAgATgAgggCTTTCT gAgTgTgggg TCAAATAgTT TCATgTTCCC 401 AAATggCCCA AAACTgACAgTTTAAACgCT gTCTTggAAC CTAATATgAC 451 AAAAgCgTgA TCTCATCCAA gATgAACTAAgTTTggTTCg TTgAAATgCT 501 AACggCCAgT TggTCAAAAA gAAACTTCCA AAAgTCgCCATACCgTTTgT 551 CTTgTTTggT ATTgATTgAC gAATgCTCAA AAATAATCTC ATTAATgCTT601 AgCgCAgTCT CTCTATCgCT TCTgAACCCg gTggCACCTg TgCCgAAACg 651CAAATggggA AACAACCCgC TTTTTggATg ATTATgCATT gTCCTCCACA 701 TTgTATgCTTCCAAgATTCT ggTgggAATA CTgCTgATAg CCTAACgTTC 751 ATgATCAAAA TTTAACTgTTCTAACCCCTA CTTgACAggC AATATATAAA 801 CAgAAggAAg CTgCCCTgTC TTAAACCTTTTTTTTTATCA TCATTATTAg 851 CTTACTTTCA TAATTgCgAC TggTTCCAAT TgACAAgCTTTTgATTTTAA 901 CgACTTTTAA CgACAACTTg AgAAgATCAA AAAACAACTA ATTATTCgAA                                                 BstBI 951ACgAggAATT CgCCTTAgAC ATgACTgTTC CTCAgTTCAA gTTgggCATT       EcoRI 1001ACgAgAAgAC CggTCTTgCT AgATTCTAAT CAAgAggATg TCAgAATgCC 1051 ATTTgCCTgAgAgATgCAgg CTTCATTTTT gATACTTTTT TATTTgTAAC 1101 CTATATAgTA TAggATTTTTTTTgTCATTT TgTTTCTTCT CgTACgAgCT 1151 TgCTCCTgAT CAgCCTATCT CgCAgCTgATgAATATCTTg TggTAggggT 1201 TTgggAAAAT CATTCgAgTT TgATgTTTTT CTTggTATTTCCCACTCCTC 1251 TTCAgAgTAC AgAAgATTAA gTgAgAAgTT CgTTTgTgCA AgCTTATCgA1301 TAAgCTTTAA TgCggTAgTT TATCACAgTT AAATTgCTAA CgCAgTCAgg 1351CACCgTgTAT gAAATCTAAC AATgCgCTCA TCgTCATCCT CggCACCgTC 1401 ACCCTggATgCTgTAggCAT AggCTTggTT ATgCCggTAc TgCCgggCCT 1451 CTTgCgggAT ATCgTCCATTCCgACAgCAT CgCCAgTCAC TATggCgTgC 1501 TgCTAgCgCT ATATgCgTTg ATgCAATTTCTATgCgCACC CgTTCTCggA 1551 gCACTgTCCg ACCgCTTTgg CCgCCgCCCA gTCCTgCTCgCTTCgCTACT 1601 TggAgCCACT ATCgACTACg CgATCATggC gACCACACCC gTCCTgTggA1651 TCTATCgAAT CTAAATgTAA gTTAAAATCT CTAPATAATT AAATAAgTCC 1701CAgTTTCTCC ATACgAACCT TAACAgCATT gCggTgAgCA TCTAgACCTT 1751 CAACAgCAgCCAgATCCATC ACTgCTTggC CAATATgTTT CAgTCCCTCA 1801 ggAgTTACgT CTTgTgAAgTgATgAACTTC TggAAggTTg CAgTgTTAAC 1851 TCCgCTgTAT TgACgggCAT ATCCgTACgTTggCAAAgTg TggTTggTAC 1901 CggAggAgTA ATCTCCACAA CTCTCTggAg AgTAggCACCAACAAACACA 1951 gATCCAgCgT gTTgTACTTg ATCAACATAA gAAgAAgCAT TCTCgATTTg2001 CAggATCAAg TgTTCAggAg CgTACTgATT ggACATTTCC AAAgCCTgCT 2051CgTAggTTgC AACCgATAgg gTTgTAgAgT gTgCAATACA CTTgCgTACA 2101 ATTTCAACCCTTggCAACTg CACAgCTTgg TTgTgAACAg CATCTTCAAT 2151 TCTggCAAgC TCCTTgTCTgTCATATCgAC AgCCAACAgA ATCACCTggg 2201 AATCAATACC ATgTTCAgCT TgAgCAgAAggTCTgAggCA ACgAAATCTg 2251 gATCAgCgTA TTTATCAgCA ATAACTAgAA CTTCAgAAggCCCAgCAggC 2301 ATgTCAATAC TACACAgggC TgATgTgTCA TTTTgAACCA TCATCTTggC2351 AgCAgTAACg AACTggTTTC CTggACCAAA TATTTTgTCA CACTTAggAA 2401CAgTTTCTgT TCCgTAAgCC ATAgCAgCTA CTgCCTgggC gCCTCCTgCT 2451 AgCACgATACACTTAgCACC AACCTTgTgg gCAACgTAgA TgACTTCTgg 2501 ggTAAgggTA CCATCCTTCTTAggTggAgA TgCAAAAACA ATTTCTTTgC 2551 AACCAgCAAC TTTggCAggA ACACCCAgCATCAgggAAgT ggAAggCAgA 2601 ATTgCggTTC CACCAggAAT ATAgAggCCA ACTTTCTCAATAggTCTTgC 2651 AAAACgAgAg CAgACTACAC CAgggCAAgT CTCAACTTgC AACgTCTCCg2701 TTAgTTgAgC TTCATggAAT TTCCTgACgT TATCTATAgA gAgATCAATg 2751gCTCTCTTAA CgTTATCTgg CAATTgCATA AgTTCCTCTg ggAAAggAgC 2801 TTCTAACACA9gTgTCTTCA AAgCgACTCC ATCAAACTTg gCAgTTAgTT 2851 CTAAAAgggC TTTgTCACCATTTTgAC9AA CATTgTCgAC AATTggTTTg 2901 ACTAATTCCA TAATCTgTTC CgTTTTCTggATAggACgAC gAAgggCATC 2951 TTCAATTTCT TgTgAggAgg CCTTAgAAAC gTCAATTTTgCACAATTCAA 3001 TACgACCTTC AgAAgggACT TCTTTAggTT TggATTCTTC TTTAggTTgT3051 TCCTTggTgT ATCCTggCTT ggCATCTCCT TTCCTTCTAg TgACCTTTAg 3101ggACTTCATA TCCAggTTTC TCTCCACCTC gTCCAACgTC ACACCgTACT 3151 TggCACATCTAACTAATgCA AAATAAAATA AgTCAgCACA TTCCCAggCT 3201 ATATCTTCCT TggATTTAgCTTCTgCAAgT TCATCAgCTT CCTCCCTAAT 3251 TTTAgCgTTC AACAAAACTT CgTCgTCAAATAACCgTTTg gTATAAgAAC 3301 CTTCTggAgC ATTgCTCTTA CgATCCCACA AggTgCTTCCATggCTCTAA 3351 gACCCTTTgA TTggCCAAAA CAggAAgTgC gTTCCAAgTg ACAgAAACCA3401 ACACCTgTTT gTTCAACCAC AAATTTCAAg CAgTCTCCAT CACAATCCAA 3451TTCgATACCC AgCAACTTTT gAgTTCgTCC AgATgTAgCA CCTTTATACC 3501 ACAAACCgTgACgACgAgAT TggTAgACTC CAgTTTgTgT CCTTATAgCC 3551 TCCggAATAg ACTTTTTggACgAgTACACC AggCCCAACg AgTAATTAgA 3601 AgAgTCAgCC ACCAAAgTAg TgAATAgACCATCggggCgg TCAgTAgTCA 3651 AAgACgCCAA CAAAATTTCA CTgACAgggA ACTTTTTgACATCTTCAgAA 3701 AgTTCgTATT CAgTAgTCAA TTgCCgAgCA TCAATAATgg ggATTATACC3751 AgAAgCAACA gTggAAgTCA CATCTACCAA CTTTgCggTC TCAgAAAAAg 3801CATAAACAgT TCTACTACCg CCATTAgTgA AACTTTTCAA ATCgCCCAgT 3851 ggAgAAgAAAAAggCACAgC gATACTAgCA TTAgCgggCA AggATgCAAC 3901 TTTATCAACC AgggTCCTATAgATAACCCT AgCgCCTggg ATCATCCTTT 3951 ggACAACTCT TTCTgCCAAA TCTAggTCCAAAATCACTTC ATTgATACCA 4001 TTATACggAT gACTCAACTT gCACATTAAC TTgAAgCTCAgTCgATTgAg 4051 TgAACTTgAT CAggTTgTgC AgCTggTCAg CAgCATAggg AAACACggCT4101 TTTCCTACCA AACTCAAggA ATTATCAAAC TCTgCAACAC TTgCgTATgC 4151AggTAgCAAg ggAAATgTCA TACTTgAAgT CggACAgTgA gTgTAgTCTT 4201 gAgAAATTCTgAAgCCgTAT TTTTATTATC AgTgAgTCAg TCATCAggAg 4251 ATCCTCTACg CCggACgCATCgTggCCggc ATCACCggCg CCACAggTgC 4301 ggTTgCTggc gCCTATATCg CCgACATCACCgATggggAA gATCgggCTC 4351 gCCACTTCgg gCTCATgAgC gCTTgTTTCg gCgTgggTATggTggCAggC 4401 CCCgTggCCg ggggACTgTT gggCgCCATC TCCTTgCATg CACCATTCCT4451 TgCggCggcg gTgCTCAACg gCCTCAACCT ACTACTgggC TgCTTCCTAA 4501TgCAggAgTC gCATAAgggA gAgCgTCgAg TATCTATgAT TggAAgTATg 4551 ggAATggTgATACCCgCATT CTTCAgTgTC TTgAggTCTC CTATCAgATT 4601 ATgCCCAACT AAAgCAACCggAggAggAgA TTTCATggTA AATTTCTCTg 4651 ACTTTTggTC ATCAgTAgAC TCgAACTgTgAgACTATCTC ggTTATgACA 4701 gCAgAAATgT CCTTCTTggA gACAgTAAAT gAAgTCCCACCAATAAAgAA 4751 ATCCTTgTTA TCAggAACAA ACTTCTTgTT TCgAACTTTT TCggTgCCTT4801 gAACTATAAA ATgTAgAgTg gATATgTCgg gTAggAATgg AgCgggCAAA 4851TgCTTACCTT CTggACCTTC AAgAggTATg TAgggTTTgT AgATACTgAT 4901 gCCAACTTCAgTgACAACgT TgCTATTTCg TTCAAACCAT TCCgAATCCA 4951 gAgAAATCAA AgTTgTTTgTCTACTATTgA TCCAAgCCAg TgCggTCTTg 5001 AAACTgACAA TAgTgTgCTC gTgTTTTgAggTCATCTTTg TATgAATAAA 5051 TCTAgTCTTT gATCTAAATA ATCTTgACgA gCCAAggCgATAAATACCCA 5101 AATCTAAAAC TCTTTTAAAA CgTTAAAAgg ACAAgTATgT CTgCCTgTAT5151 TAAACCCCAA ATCAgCTCgT AgTCTgATCC TCATCAACTT gAggggCACT 5201ATCTTgTTTT AgAgAAATTT gCggAgATgC gATATCgAgA AAAAggTACg 5251 CTgATTTTAAACgTgAAATT TATCTCAAgA TCgCggCCgC gATCTCgAAT 5301 AATAACTgTT ATTTTTCAgTgTTCCCgATC TgCgTCTATT TCACAATACC 5351 AACATgAgTC AgCTTATCgA TgATAAgCTgTCAAACATgA gAATTAATTC 5401 gATgATAAgC TgTCA~ACAT gAgAAATCTT gAAgACgAAAgggCCTCgTg 5451 ATACgCCTAT TTTTATAggT TAATgTCATg ATAATAATgg TTTCTTAgAC5501 gTCAggTggC ACTTTTCggg gAAATgTgCg CggAACCCCT ATTTgTTTAT 5551TTTTCTAAAT ACATTCAAAT ATgTATCCgC TCATgAgACA ATAACCCTgA 5601 TAAATgCTTCAATAATATTg AAAAAggAAg AgTATgAgTA TTCAACATTT 5651 CCgTgTCgCC CTTATTCCCTTTTTTgCggC ATTTTgCCTT CCTgTTTTTg 5701 CTCACCCAgA AACgCTggTg AAAgTAAAAgATgCTgAAgA TCAgTTgggT 5751 gCACgAgTgg gTTACATCgA ACTggATCTC AACAgCggTAAgATCCTTgA 5801 gAgTTTTCgC CCCgAAgAAC gTTTTCCAAT gATgAgCACT TTTAAAgTTC5851 TgCTATgTgg CgCggTATTA TCCCgTgTTg ACgCCgggCA AgAgCAACTC 5901ggTCgCCgCA TACACTATTC TCAgAATgAC TTggTTgAgT ACTCACCAgT 5951 CACAgAAAAgCATCTTACgg ATggCATgAC AgTAAgAgAA TTATgCAgTg 6001 CTgCCATAAC CATgAgTgATAACACTgCgg CCAACTTACT TCTgACAACg 6051 ATCggAggAC CgAAggAgCT AACCgCTTTTTTgCACAACA TgggggATCA 6101 TgTAACTCgC CTTgATCgTT gggAACCggA gCTgAATgAAgCCATACCAA 6151 ACgACgAgCg TgACACCACg ATgCCTgCAg CAATggCAAC AACgTTgCgC6201 AAACTATTAA CTggCgAACT ACTTACTCTA gCTTCCCggC AACAATTAAT 6251AgACTggATg gAggCggATA AAgTTgCAgg ACCACTTCTg CgCTCggCCC 6301 TTCCggCTggCTggTTTATT gCTgATAAAT CTggAgCCgg TgAgCgTggg 6351 TCTCgCggTA TCATTgCAgCACTggggCCA gATggTAAgC CCTCCCgTAT 6401 CgTAgTTATC TACACgACgg ggAgTCAggCAACTATqgAT gAACgAAATA 6451 gACAgATCgC TgAgATAggT gCCTCACTgA TTAAgCATTggTAACTgTCA 6501 gACCAAgTTT ACTCATATAT ACTTTAgATT gATTTAAATT gTAAACgTTA6551 ATATTTTgTT AAAATTCgCg TTAAATTTTT gTTAAATCAg CTCATTTTTT 6601AACCAATAgg CCgAAATCgg CAAAATCCCT TATAAATCAA AAgAATAgAC 6651 CgAgATAgggTTgAgTgTTg TTCCAgTTTg gAACAAgAgT CCACTATTAA 6701 AgAACgTggA CTCCAACgTCAAAgggCgAA AAACCgTCTA TCAgggCgAT 6751 ggCCCACTAC gTgAACCATC ACCCTAATCAAgTTTTTTgg ggTCgAggTg 6801 CCgTAAAgCA CTAAATCggA ACCCTAAAgg gAgCCCCCgATTTAgAgCTT 6851 gACggggAAA gCCggCgAAC gTggCgAgAA AggAAgggAA gAAAgCgAAA6901 ggAgCgggCg CTAgggCgCT ggCAAgTgTA gCggTCACgC TgCgCgTAAC 6951CACCACACCC gCCgCgCTTA ATgCgCCgCT ACAgggCgCg TAAAAggATC 7001 TAggTgAAgATCCTTTTTgA TAATCTCATg ACCAAAATCC CTTAACgTgA 7051 gTTTTCgTTC CACTgAgCgTCAgACCCCgT AgAAAAgATC AAAggATCTT 7101 CTTgAgATCC TTTTTTTCTg CgCgTAATCTgCTgCTTgCA AACAAAAAAA 7151 CCACCgCTAC CAgCggTggT TTgTTTgCCg gATCAAgAgCTACCAACTCT 7201 TTTTCCgAAg gTAACTggCT TCAgCAgAgC gCAgATACCA AATACTgTCC7251 TTCTA9TgTA gCCgTAgTTA ggCCACCACT TCAAgAACTC TgTAgCACCg 7301CCTACATACC TCgCTCTgCT AATCCTgTTA CCAgTggCTg CTgCCAgTgg 7351 CgATAAgTCgTgTCTTACCg ggTTggACTC AAgACgATAg TTACCggATA 7401 AggCgCAgCg gTCgggCTgAACggggggTT CgTgCACACA gCCCAgCTTg 7451 gAgCgAACgA CCTACACCgA ACTgAgATACCTACAgCgTg AgCATTgAgA 7501 AAgCgCCACg CTTCCCgAAg ggAgAAAggC ggACAggTATCCggTAAgCg 7551 gCAgggTCgg AACAggAgAg CgCACgAggg AgCTTCCAgg gggAAACgCC7601 TggTATCTTT ATAgTCCTgT CgggTTTCgC CACCTCTgAC TTgAgCgTCg 7651ATTTTTgTgA TgCTCgTCAg gggggCggAg CCTATggAAA AACgCCAgCA 7701 ACgCggCCTTTTTACggTTC CTggCCTTTT gCTggCCTTT TgCTCACATg 7751 TTCTTTCCTg CgTTATCCCCTgATTCTgTg gATAACCgTA TTACCgCCTT 7801 TgAgTgAgCT gATACCgCTC gCCgCAgCCgAACgACCgAg CgCAgCgAgT 7851 CAgTgAgCgA ggAAgCggAA gAgCgCCTgA TgCggTATTTTCTCCTTACg 7901 CATCTgTgCg gTATTTCACA CCgCATATgg TgCACTCTCA gTACAATCTg7951 CTCTgATgCC gCATAgTTAA gCCAgTATAC ACTCCgCTAT CgCTACgTgA 8001CTgggTCATg gCTgCgCCCC gACACCCgCC AACACCCgCT gACgCgCCCT 8051 gACgggCTTgTCTgCTCCCg gCATCCgCTT ACAgACAAgC TgTgACCgTC 8101 TCCgggAgCT gCATgTgTCAgAggTTTTCA CCgTCATCAC CgAAACgCgC 8151 gAggCAg

[0252] TABLE 251 pHIL-D2(MFαPrePro::EPI-HNE-3) 8584 b.p. DNA has SEQ IDNO. 071; Encoded polypeptide has SEQ ID NO. 072. DNA is circular anddouble stranded, only one strand is shown. Translation of the protein tobe expressed is shown.         1          2          3          4          5 12345678901234567890 1234567890 1234567890 1234567890 1 AgATCgCggC CgCgATCTAACATCCAAAgA CgAAAggTTg AATgAAACCT 51 TTTTgCCATC CgACATCCAC AggTCCATTCTCACACATAA gTgCCAAACg 101 CAACAggAgg ggATACACTA gCAgCAgACC gTTgCAAACgCAggACCTCC 151 ACTCCTCTTC TCCTCAACAC CCACTTTTgC CATCgAAAAA CCAgCCCAgT201 TATTgggCTT gATTggAgCT CgCTCATTCC AATTCCTTCT ATTAggCTAC 251TAACACCATg ACTTTATTAg CCTgTCTATC CTggCCCCCC TggCgAggTC 301 ATgTTTgTTTATTTCCgAAT gCAACAAgCT CCgCATTACA CCC9AACATC 351 ACTCCAgATg AgggCTTTCTgAgTgTgggg TCAAATAgTT TCATgTTCCC 401 AAATggCCCA AAACTgACAg TTTAAACgCTgTCTTggAAC CTAATATgAC 451 AAAAgCgTgA TCTCATCCAA gATgAACTAA gTTTggTTCgTTgAAATgCT 501 AACggCCAgT TggTCAAAAA 9AAACTTCCA AAAgTCgCCA TACCgTTTgT551 CTTgTTTggT ATTgATTgAC gAATgCTCAA AAATAATCTC ATTAATgCTT 601AgCgCAgTCT CTCTATCgCT TCTgAACCCg gTggCACCTg TgCCgAAACg 651 CAAATggggAAACAACCCgC TTTTTggATg ATTATgCATT gTCCTCCACA 701 TTgTATgCTT CCAAgATTCTggTgggAATA CTgCTgATAg CCTAACgTTC 751 ATgATCAAAA TTTAACTgTT CTAACCCCTACTTgACAggC AATATATAAA 801 CAgAAggAAg CTgCCCTgTC TTAAACCTTT TTTTTTATCATCATTATTAg 851 CTTACTTTCA TAATTgCgAC TggTTCCAAT TgACAAgCTT TTgATTTTAA901 CgACTTTTAA CgACAACTTg AgAAgATCAA AAAACAACTA ATTA TTCgAA !                                                 BstBI ACg ! M   R   F   P   S   I   F   T   A   V   L   F   A  13 ATg AgA TTCCCA TCT ATC TTC ACT gCT gTT TTg TTC gCT !      |  BsaBI    | ! ! A   S   S   A   L   A   A   P   V   N   T   T   T   E  27 gCT TCC TCTgCT TTg gCT gCT CCA gTT AAC ACC ACT ACT gAA !                          BpmI   HpaI               BbsI ! ! D   E   T   A   Q   I   P   A   E   A   V   I   G   Y  41 gAC gAg ACTgCT CAA ATT CCT gCT gAg gCT gTC ATC ggT TAC ! BbsI ! ! S   D   L   E   G   D   F   D   V   A   V   L   P   F  55 TCT gAC TTggAA ggT gAC TTC gAC gTC gCT gTT TTg CCA TTC !                             AatII ! ! S   N   S   T   N   N   G   L   L   F   I   N   T   T  69 TCT AAC TCTACT AAC AAC ggT TTg TTg TTC ATC AAC ACT ACC ! ! I   A   S   I   A   A   K   E   E   G   V   S   L   D  83 ATC gCT TCTATC gCT gCT AAg gAg gAA ggT gTT TCC TTg gAC ! ! K   R       A   A   C   N   L   P  91 AAg AgA  |  gCT gCT TgT AAC TTgCCA           ----- Site of cleavage ! ! I   V   R   G   P   C   I   A   F   F   P   R   W   A 105 ATC gTC AgAggT CCA TgC ATT gCT TTC TTC CCA AgA Tgg gCT !                     NsiI ! F   D   A   V   K   G   K   C   V   L   F   P   Y   G 119 TTC gAC gCTgTT AAg ggT AAg TgC gTC TTg TTC CCA TAC ggT !                                           |  PflMI ! G   C   Q   G   N   G   N   K   F   Y   S   E   K   E 133 ggT TgT CAAggT AAC ggT AAC AAg TTC TAC TCT gAg AAg gAg ! PflMI ! C   R   E   Y   C   G   V   P 141 TgT AgA gAg TAC TgT ggT gTT CCA TAgTAA gAATTCgCCT !                                       EcoRI                                              TAgACATg 1401 ACTgTTCCTCAgTTCAAgTT gggCATTACg AgAAgACCgg TCTTgCTAgA 1451 TTCTAATCAA gAggATgTCAgAATgCCATT TgCCTgAgAg ATgCAggCTT 1501 CATTTTTgAT ACTTTTTTAT TTgTAACCTATATAgTATAg gATTTTTTTT 1551 gTCATTTTgT TTCTTCTCgT ACgAgCTTgC TCCTgATCAgCCTATCTCgC 1601 AgCTgATgAA TATCTTgTgg TAggggTTTg ggAAAATCAT TCgAgTTTgA1651 TgTTTTTCTT ggTATTTCCC ACTCCTCTTC AgAgTACAgA AgATTAAgTg 1701AgAAgTTCgT TTgTgCAAgC TTATCgATAA gCTTTAATgC ggTAgTTTAT 1751 CACAgTTAAATTgCTAACgC AgTCAggCAC CgTgTATgAA ATCTAACAAT 1801 gCgCTCATCg TCATCCTCggCACCgTCACC CTggATgCTg TAggCATAgg 1851 CTTggTTATg CCggTACTgC CgggCCTCTTgCgggATATC gTCCATTCCg 1901 ACAgCATCgC CAgTCACTAT ggCgTgCTgC TAgCgCTATATgCgTTgATg 1951 CAATTTCTAT gCgCACCCgT TCTCggAgCA CTgTCCgACC gCTTTggCCg2001 CCgCCCAgTC CTgCTCgCTT CgCTACTTgg AgCCACTATC gACTACgCgA 2051TCATggCgAC CACACCCgTC CTgTggATCT ATCgAATCTA AATgTAAgTT 2101 AAAATCTCTAAATAATTAAA TAAgTCCCAg TTTCTCCATA CgAACCTTAA 2151 CAgCATTgCg gTgAgCATCTAgACCTTCAA CAgCAgCCAg ATCCATCACT 2201 gCTTggCCAA TATgTTTCAg TCCCTCAggAgTTACgTCTT gTgAAgTgAT 2251 gAACTTCTgg AAggTTgCAg TgTTAACTCC gCTgTATTgACgggCATATC 2301 CgTACgTTgg CAAAgTgTgg TTggTACCgg AggAgTAATC TCCACAACTC2351 TCTggAgAgT AggCACCAAC AAACACAgAT CCAgCgTgTT gTACTTgATC 2401AACATAAgAA gAAgCATTCT CgATTTgCAg gATCAAgTgT TCAggAgCgT 2451 ACTgATTggACATTTCCAAA gCCTgCTCgT AggTTgCAAC CgATAgggTT 2501 gTAgAgTgTg CAATACACTTgCgTACAATT TCAACCCTTg gCAACTgCAC 2551 AgCTTggTTg TgAACAgCAT CTTCAATTCTggCAAgCTCC TTgTCTgTCA 2601 TATCgACAgC CAACAgAATC ACCTgggAAT CAATACCATgTTCAgCTTgA 2651 gCAgAAggTc TgAggCAACg AAATCTggAT CAgCgTATTT ATCAgCAATA2701 ACTAgAACTT CAgAAggCCC AgCAggCATg TCAATACTAC ACAgggCTgA 2751TgTgTCATTT TgAACCATCA TCTTggCAgC AgTAACgAAC TggTTTCCTg 2801 9ACCAAATATTTTgTCACAC TTAggAACAg TTTCTgTTCC gTAAgCCATA 2851 gCAgCTACTg CCTgggCgCCTCCTgCTAgC ACgATACACT TAgCACCAAC 2901 CTTgTgggCA ACgTAgATgA CTTCTgqggTAAgggTACCA TCCTTCTTAg 2951 gTggAgATgC AAAAACAATT TCTTTgCAAC CAgCAACTTTggCAggAACA 3001 CCCAgCATCA gggAAgTggA AggCA9AATT gCggTTCCAC CAggAATATA3051 gAggCCAACT TTCTCAATAg gTCTTgCAAA ACgAgAgCAg ACTACACCAg 3101ggCAAgTCTC AACTTgCAAC gTCTCCgTTA gTTgAgCTTC ATggAATTTc 3151 CTgACgTTATCTATAgAgAg ATCAATggCT CTCTTAACgT TATCTggCAA 3201 TTgCATAAgT TCCTCTgggAAAggAgCTTC TAACACAggT gTCTTCAAAg 3251 CgACTCCATC AAACTTggCA gTTAgTTCTAAAAgggCTTT gTCACCATTT 3301 TgACgAACAT TgTCgACAAT TggTTTgACT AATTCCATAATCTgTTCCgT 3351 TTTCTggATA ggACgACgAA gggCATCTTC AATTTCTTgT gAggAggCCT3401 TAgAAACgTC AATTTTgCAC AATTCAATAC gACCTTCAgA AgggACTTCT 3451TTAggTTTgg ATTCTTCTTT AggTTgTTcC TTggTgTATC CTggCTTggC 3501 ATCTCCTTTCCTTCTAgTgA CCTTTAgggA CTTCATATCC AggTTTCTCT 3551 CCACCTCgTC CAACgTCACACCgTACTTgg CACATCTAAC TAATgCAAAA 3601 TAAAATAAgT CAgCACATTC CCAggCTATATCTTCCTTgg ATTTAgCTTC 3651 TgCAAgTTCA TCAgCTTCCT CCCTAATTTT AgCgTTCAACAAAACTTCgT 3701 CgTCAAATAA CCgTTTggTA TAAgAACCTT CTggAgCATT gCTCTTACgA3751 TCCCACAAgg TgCTTCCATg gCTCTAAgAC CCTTTgATTg gCCAAAACAg 3801gAAgTgCgTT CCAAgTgACA gAAACCAACA CCTgTTTgTT CAACCACAAA 3851 TTTCAAgCAgTCTCCATCAC AATCCAATTC gATACCCAgC AACTTTTgAg 3901 TTCgTCCAgA TgTAgCACCTTTATACCACA AACCgTgACg ACgAgATTgg 3951 TAgACTCCAg TTTgTgTCCT TATAgCCTCCggAATAgACT TTTTggACgA 4001 gTACACCAgg CCCAACgAgT AATTAgAAgA gTCAgCCACCAAAgTAgTgA 4051 ATAgACCATC ggggCggTCA gTAgTCAAAg ACgCCAACAA AATTTCACTg4101 ACAgggAACT TTTTgACATC TTCAgAAAgT TCgTATTCAg TAgTCAATTg 4151CCgAgCATCA ATAATggggA TTATACCAgA AgCAACAgTg gAAgTCACAT 4201 CTACCAACTTTgCggTCTCA gAAAAAgCAT AAACAgTTCT ACTACCgCCA 4251 TTAgTgAAAC TTTTCAAATCgCCCAgTggA gAAgAAAAAg gCACAgCgAT 4301 ACTAgCATTA gCgggCAkgg ATgCAACTTTATCAACCAgg gTCCTATAgA 4351 TAACCCTAgC gCCTgggATC ATCCTTTggA CAACTCTTTCTgCCAAATCT 4401 AggTCCAAAA TCACTTCATT gATACCATTA TACggATgAC TCAACTTgCA4451 CATTAACTTg AAgCTCAgTC gATTgAgTgA ACTTgATCAg gTTgTgCAgC 4501TggTCAgCAg CATAgggAAA CACggCTTTT CCTACCAAAC TCAAggAATT 4551 ATCAAACTCTgCAACACTTg CgTATgCAgg TAgCAAgggA AATgTCATAC 4601 TTgAAgTCgg ACAgTgAgTgTAgTCTTgAg AAATTCTgAA gCCgTATTTT 4651 TATTATCAgT gAgTCAgTCA TCAggAgATCCTCTACgCCg gACgCATCgT 4701 ggCCggCATC ACCggCgCCA CAggTgCggT TgCTggCgCCTATATCgCCg 4751 ACATCACCgA TggggAAgAT CgggCTCgCC ACTTCgggCT CATgAgCgCT4801 TgTTTCggCg TgggTATggT ggCAggCCCC gTggCCgggg gACTgTTggg 4851CgCCATCTCC TTgCATgCAC CATTCCTTgC ggCggCggTg CTCAACggCC 4901 TCAACCTACTACTgggCTgC TTCCTAATgC AggAgTCgCA TAAgggAgAg 4951 CgTCgAgTAT CTATgATTggAAgTATgggA ATggTgATAC CCgCATTCTT 5001 CAgTgTCTTg AggTCTCCTA TCAgATTATgCCCAACTAAA gCAACCggAg 5051 gAggAgATTT CATggTAAAT TTCTCTgACT TTTggTCATCAgTAgACTCg 5101 AACTgTgAgA CTATCTCggT TATgACAgCA gAAATgTCCT TCTTggAgAC5151 AgTAAATgAA gTCCCACCAA TAAAgAAATC CTTgTTATCA ggAACAAACT 5201TCTTgTTTCg AACTTTTTCg gTgCCTTgAA CTATAAAATg TAgAgTggAT        BstBI 5251ATgTCgggTA ggAATggAgC gggCAAATgC TTACCTTCTg gACCTTCAAg 5301 AggTATgTAgggTTTgTAgA TACTgATgCC AACTTCAgTg ACAACgTTgC 5351 TATTTCgTTC AAACCATTCCgAATCCAgAg AAATCAAAgT TgTTTgTCTA 5401 CTATTgATCC AAgCCAgTgC ggTCTTgAAACTgACAATAg TgTgCTCgTg 5451 TTTTgAg9TC ATCTTTgTAT gAATAAATCT AgTCTTTgATCTAAATAATC 5501 TTgACgAgCC AAggCgATAA ATACCCAAAT CTAAAACTCT TTTAAAACgT5551 TAAAAggACA AgTATgTCTg CCTgTATTAA ACCCCAAATC AgCTCgTAgT 5601CTgATCCTCA TCAACTT9Ag gggCACTATC TTgTTTTAgA gAAATTTgCg 5651 gAgATgCgATATCgAgAAAA AggTACgCTg ATTTTAAACg TgAAATTTAT 5701 CTCAAgATCg CggCCgCgATCTCgAATAAT AACTgTTATT TTTCAgTgTT 5751 CCCgATCTgC gTCTATTTCA CAATACCAACATgAgTCAgC TTATCgATgA 5801 TAAgCTgTCA AACATgAgAA TTAATTC9AT gATAAgCTgTCAAACATgAg 5851 AAATCTTgAA gACgAAAggg CCTCgTgATA CgCCTATTTT TATAggTTAA5901 TgTCATgATA ATAATggTTT CTTAgACgTC AggTggCACT TTTCggggAA                           AatII 5951 ATgTgCgCgg AACCCCTATT TgTTTATTTTTCTAAATACA TTCAAATATg 6001 TATCCgCTCA TgAgACAATA ACCCTgATAA ATgCTTCAATAATATTgAAA 6051 AAggAAgAgT ATgAgTATTC AACATTTCCg TgTCgCCCTT ATTCCCTTTT6101 TTgCggCATT TTgCCTTCCT gTTTTTgCTC ACCCAgAAAC gCTggTgAAA 6151gTAAAAgATg CTgAAgATCA gTTgggTgCA CgAgTgggTT ACATCgAACT 6201 ggATCTCAACAgCggTAAgA TCCTTgAgAg TTTTCgCCCC gAAgAACgTT 6251 TTCCAATgAT gAgCACTTTTAAAgTTCTgC TATgTggCgC ggTATTATCC 6301 CgTgTTgACg CCgggCAAgA gCAACTCggTCgCCgCATAC ACTATTCTCA 6351 gAATgACTTg gTTgAgTACT CACCAgTCAC AgAAAAgCATCTTACggATg 6401 gCATgACAgT AAgAgAATTA TgCAgTgCTg CCATAACCAT gAgTgATAAC6451 ACTgCggCCA ACTTACTTCT gACAACgATC ggAggACCgA AggAgCTAAC 6501CgCTTTTTTg CACAACATgg gggATCATgT AACTCgCCTT gATCgTTggg 6551 AACCggAgCTgAATgAAgCC ATACCAAACg ACgAgCgTgA CACCACgATg 6601 CCTgCAgCAA TggCAACAACgTTgCgCAAA CTATTAACTg gCgAACTACT 6651 TACTCTA9CT TCCCggCAAC AATTAATAgACTggATggAg gCggATAAAg 6701 TTgCAggACC ACTTCTgCgC TCggCCCTTC CggCTggCTggTTTATTgCT 6751 gATAAATCTg gAgCCggTgA gCgTgggTCT CgCggTATCA TTgCAgCACT6801 ggggCCAgAT ggTAAgCCCT CCCgTATCgT AgTTATCTAC ACgACggggA 6851gTCAggCAAC TATggATgAA CgAAATAgAC AgATCgCTgA gATAggTgCC 6901 TCACTgATTAAgCATTggTA ACTgTCAgAC CAAgTTTACT CATATATACT 6951 TTAgATTgAT TTAAATTgTAAACgTTAATA TTTTgTTAAA ATTCgCgTTA 7001 AATTTTTgTT AAATCAgCTC ATTTTTTAACCAATAggCCg AAATCggCAA 7051 AATCCCTTAT AAATCAAAAg AATAgACCgA gATAgggTTgAgTgTTgTTC 7101 CAgTTTggAA CAAgAgTCCA CTATTAAAgA ACgTggACTC CAACgTCAAA7151 gggCgAAAAA CCgTCTATCA gggCgATggC CCACTACgTg AACCATCACC 7201CTAATCAAgT TTTTTggggT CgAggTgCCg TAAAgCACTA AATCggAACC 7251 CTAAAgggAgCCCCCgATTT AgAgCTTgAC ggggAAAgCC ggCgAACgTg 7301 gCgAgAAAgg AAgggAAgAAAgCgAAAggA gCgggCgCTA gggCgCTggC 7351 AAgTgTAgCg gTCACgCTgC gCgTAACCACCACACCCgCC gCgCTTAATg 7401 CgCCgCTACA gggCgCgTAA AAggATCTAg gTgAAgATCCTTTTTgATAA 7451 TCTCATgACC AAAATCCCTT AACgTgAgTT TTCgTTCCAC TgAgCgTCAg7501 ACCCCgTAgA AAAgATCAAA ggATCTTCTT gAgATCCTTT TTTTCTgCgC 7551gTAATCTgCT gCTTgCAAAC AAAAAAACCA CCgCTACCAg CggTggTTTg 7601 TTTgCCggATCAAgAgCTAC CAACTCTTTT TCCgAAggTA ACTggCTTCA 7651 gCAgAgCgCA gATACCAAATACTgTCCTTC TAgTgTAgCC gTAgTTAggC 7701 CACCACTTCA AgAACTCTgT AgCACCgCCTACATACCTCg CTCTgCTAAT 7751 CCTgTTACCA gTggCTgCTg CCAgTggCgA TAAgTCgTgTCTTACCgggT 7801 TggACTCAAg ACgATAgTTA CCggATAAgg CgCAgCggTC gggCTgAACg7851 gggggTTCgT gCACACAgCC CAgCTTggAg CgAACgACCT ACACCgAACT 7901gAgATACCTA CAgCgTgAgC ATTgAgAAAg CgCCACgCTT CCCgAAgggA 7951 gAAAggCggACAggTATCCg gTAAgCggCA gggTCggAAC AggAgAgCgC 8001 ACgAgggAgC TTCCAgggggAAACgCCTgg TATCTTTATA gTCCTgTCgg 8051 gTTTCgCCAC CTCTgACTTg AgCgTCgATTTTTgTgATgC TCgTCAgggg 8101 ggCggAgCCT ATggAAAAAC gCCAgCAACg CggCCTTTTTACggTTCCTg 8151 gCCTTTTgCT ggCCTTTTgC TCACATgTTC TTTCCTgCgT TATCCCCTgA8201 TTCTgTggAT AACCgTATTA CCgCCTTTgA gTgAgCTgAT ACCgCTCgCC 8251gCAgCCgAAC gACCgAgCgC AgCgAgTCAg TgAgCgAggA AgCggAAgAg 8301 CgCCTgATgCggTATTTTCT CCTTACgCAT CTgTgCggTA TTTCACACCg 8351 CATATggTgC ACTCTCAgTACAATCTgCTC TgATgCCgCA TAgTTAAgCC 8401 AgTATACACT CCgCTATCgC TACgTgACTgggTCATggCT gCgCCCCgAC 8451 ACCCgCCAAC ACCCgCTgAC gCgCCCTgAC gggCTTgTCTgCTCCCggCA 8501 TCCgCTTACA gACAAgCTgT gACCgTCTCC gggAgCTgCA TgTgTCAgAg8551 gTTTTCACCg TCATCACCgA AACgCgCgAg gCAg Restriction map of pHIL-D2(MFαPrePro::EPI-HNE-3) Non-cutters AflII ApaI AscI AvaI AvrII BamBIBglII BssHII BstEII MluI NruI PacI PmlI RsrII SacII SfiI SnaBI SpeI XhoIXmaI Cutters, 3 or fewer sites AatII 2 1098 5925 ApaLI 3 6176 7859 8357AflIII 1 8173 AseI 3 591 5820 6672 AgeI 1 1436 BglI 3 284 2717 6724AlwNI 3 2828 2852 7759 BsaAI 2 7185 8421 BsgI 2 2545 4494 PvuI 1 6476BsiWI 2 1568 2301 PvuII 2 1600 4497 BspDI 2 1723 5793 SacI 1 216 BspEI 13978 SalI 1 3312 BspMI 1 4576 ScaI 2 1360 6365 Bst1107I 1 8402 SphI 14863 BstBI (AsuII) 2 945 5207 SspI 3 2806 6041 6977 BstXI 3 711 27652896 StuI 1 3395 Bsu36I 1 2223 TthlllI 1 8426 DraIII 2 3754 7182 XbaI 12168 EagI 3 7 5711 8591 XcmI 1 711 Eam1105I 2 5077 6843 Ecl136I 1 216Eco47III 2 1932 4795 EcoNI 3 3433 4923 5293 EcoRI 1 1383 EcoRV 2 18855658 EspI (BsaI) 2 3120 8524 EspI (Bpu1102I) 1 597 FspI 2 1960 6623HindIII 3 885 1717 1729 HpaI 2 1017 2272 KpnI 2 2323 2934 MscI 2 22043789 NcoI 1 3766 NdeI 1 8351 NgoMI 2 4702 7288 NheI 2 1929 2875 NotI 3 65710 8590 NsiI 2 684 1241 PflMI 2 196 1302 PmeI 1 420 PpuMI 2 142 4339PstI 1 6602 Table 252: BstBI-AatII-EcoRI cassette for expression of EPI-HNE-4 DNA has SEQ ID NO. 073; amino-acid sequence has SEQ ID NO. 074 !              M   R   P   P   S   T   F   T 5′ TTCgAA  ACg ATg AgA TTCCCA TCT ATC TTC ACT    BstBI          |  BsaBI    | !          A   V   L   F   A   13          gCT gTT TTg TTC gCT ! ! A   S   S   A   L   A   A   P   V   N   T   T   T   E 27 gCT TCC TCTgCT TTg gCT gCT CCA gTT AAC ACC ACT ACT gAA !                          BpmI   HpaI               BbsI ! ! D   E   T   A   Q   I   P   A   E   A   V   I   G   Y 41 gAC gAg ACTgCT OAA ATT CCT gCT gAg gCT gTC ATC ggT TAC ! BbsI ! S   D   L   E   G   D   F   D   V   A   V   L   P   F 55 TCT gAC TTggAA ggT gAC TTC gAC gTC gCT gTT TTg CCA TTC !          AatII ! S   N   S   T   N   N   G   L   L   F   I   N   T   T 69 TCT AAC TCTACT AAC AAC ggT TTg TTg TTC ATC AAC ACT ACC ! I   A   S   I   A   A   K   E   E   G   V   S   L   D 83 ATC gCT TCTATC gCT gCT AAg gAg gAA ggT gTT TCC TTg gAC ! K   R   E   A   C   N   L   P 91 AAg AgA gAg gOT TgT AAC TTg CCA !I   V   R   G   P   C   I   A   F   F   P   R   W   A 105 ATC gTC AgAggT CCA TgC ATT gCT TTC TTC CCA AgA Tgg gCT !                     NsiI !!  F   D   A   V   K   C   K   C   V   L   F   P   Y   G 119 TTC gAC gCTgTT AAg ggT AAg TgC gTC TTg TTC CCA TAC ggT !                                           |  PflMI ! ! G   C   Q   C   N   G   N   K   F   Y   S   E   K   E 133 ggT TgT CAAggT AAC ggT AAC AAg TTC TAC TCT gAg AAg gAg ! PflMI ! !C   R   E   Y   C   G   V   P   .   . 141 TgT AgA gAg TAC TgT ggT gTTCCA TAg TAA gAATTC !                                          EcoRI

[0253] TABLE 253 pD2pick(MFαPrePro::EPI-HNE-3), 8590 bp, CIRCULAR dsDNA,one strand shown. pD2pick(MFαPrePro::EPI-HNE-3) DNA has SEQ ID NO. 075Encoded protein has SEQ ID NO. 076         1          2          3          4          5 12345678901234567890 1234567890 1234567890 1234567890 1 AgATCgCggC CgCgATCTAACATCCAAAgA CgAAAggTTg AATgAAACCT 51 TTTTgCCATC CgACATCCAC AggTCCATTCTCACACATAA gTgCCAAACg 101 CAACAggAgg ggATACACTA gCAgCAgACC gTTgCAAACgCAggACCTCC 151 ACTCCTCTTC TCCTCAACAC CCACTTTTgC CATCgAAAAA CCAgCCCAgT201 TATTgggCTT gATTg gAgCT C gCTCATTCC AATTCCTTCT ATTAggCTAC                 SacI 251 TAACACCATg ACTTTATTAg CCTgTCTATC CTggCCCCCCTggCgAggTC 301 ATgTTTgTTT ATTTCCgAAT gCAACAAgCT CCgCATTACA CCCgAACATC351 ACTCCAgATg AgggCTTTCT gAgTgTgggg TCAAATAgTT TCATgTTCCC 401AAATggCCCA AAACTgACA g TTTAAAC gCT gTCTTggAAC CTAATATgAC                       PmeI 451 AAAAgCgTgA TCTCATCCAA gATgAACTAAgTTTggTTCg TTgAAATgCT 501 AACggCCAgT TggTCAAAAA gAAACTTCCA AAAgTCgCCATACCgTTTgT 551 CTTgTTTggT ATTgATTgAC gAATgCTCAA AAATAATCTC ATTAATgCTTAgC EspI 604 gCAgTCT CTCTATCgCT TCTgAACCCg gTggCACCTg TgCCgAAACg 651CAAATggggA AACAACCCgC TTTTTggATg ATTATgCATT gTCCTCCACA 701 TTgTATgCTTCCAAgATTCT gg TgggAATA CTgCTgATAg CCTAACgTTC                XcmI 751ATgATCAAAA TTTAACTgTT CTAACCCCTA CTTgACAggC AATATATAAA 801 CAgAAggAAgCTgCCCTgTC TTAAACCTTT TTTTTTATCA TCATTATTAg 851 CTTACTTTCA TAATTgCgACTggTTCCAAT TgACAAgCTT TTgATTTTAA 901 CgACTTTTAA CgACAACTTg AgAAgATCAAAAAACAACTA ATTATTCgAA!                                                    BstBI 951 ACg ! ! M   R   F   P   S   I   F   T   A   V   L   F   A 954 ATg AgA TTC CCATCT ATC TTC ACT gCT gTT TTg TTC gCT ! ! A   S   S   A   L   A   A   P   V   N   T   T   T 993 gCT TCC TCT gCTTTg gCT gCT CCA gTT AAC ACC ACT ACT ! ! E   D   E   T   A   Q   I   P   A   E   A   V   I 1032 gAA gAC gAg ACTgCT CAA ATT CCT gCT gAg gCT gTC ATC ! ! G   Y   S   D   L   E   G   D   F   D   V   A   V 1071 ggT TAC TCT gACTTg gAA ggT gAC TTC gAC gTC  gCT gTT                                          AatII ! ! L   P   F   S   N   S   T   N   N   G   L   L   F ! 1110 TTg CCA TTCTCT AAC TCT ACT AAC AAC ggT TTg TTg TTC ! ! N   T   T   I   A   S   I   A   A   K   E   E 1149 ATC AAC ACT ACC ATCgCT TCT ATC gCT gCT AAg gAg gAA ! ! G   V   S   L   D   K   R   A   A   C   N   L   P 1188 ggT gTT TCC TTggAC AAg AgA gCT gCT TgT AAC TTg CCA ! ! I   V   R   G   P   C   I   A   F   F   P   R   W 1227 ATC gTC AgA ggTCCA TgC ATT gCT TTC TTC CCA AgA Tgg ! ! A   F   D   A   V   K   G   K   C   V   L   F   P 1266 gCT TTC gAC gCTgTT AAg ggT AAg TgC gTC TTg TTC CCA ! ! Y   G   G   C   Q   G   N   G   N   K   F   Y   S 1305 TAC ggT ggT TgTCAA ggT AAC ggT AAC AAg TTC TAC TCT ! ! E   K   E   C   R   E   Y   C   G   V   P   .   . 1344 gAg AAg gAg TgTAgA gAg TAC TgT ggT gTT CCA TAg TAA ! 1383gAATTC                                   gC CTTAgACATg !      ECoRI 1401ACTgTTCCTC AgTTCAAgTT gggCATTACg AgAAg ACCgg T CTTgCTAgA                                            AegI 1451 TTCTAATCAAgAggATgTCA gAATgCCATT TgCCTgAgAg ATgCAggCTT 1501 CATTTTTgAT ACTTTTTTATTTgTAACCTA TATAgTATAg gATTTTTTTT 1551 gTCATTTTgT TTCTTCTCgT ACgAgCTTgCTCCTgATCAg CCTATCTCgC 1601 AgCTgATgAA TATCTTgTgg TAggggTTTg ggAAAATCATTCgAgTTTgA 1651 TgTTTTTCTT ggTATTTCCC ACTCCTCTTC AgAgTACAgA AgATTAAgTg1701 AgAAgTTCgT TTgTgCAAgC TTATCgATAA gCTTTAATgC ggTAgTTTAT 1751CACAgTTAAA TTgCTAACgC AgTCAggCAC CgTgTATgAA ATCTAACAAT 1801 gCgCTCATCgTCATCCTCgg CACCgTCACC CTggATgCTg TAggCATAgg 1851 CTTggTTATg CCggTACTgCCgggCCTCTT gCgggATATC gTCCATTCCg 1901 ACAgCATCgC CAgTCACTAT ggCgTgCTgCTAgCgCTATA TgCgTTgATg 1951 CAATTTCTAT gCgCACCCgT TCTCggAgCA CTgTCCgACCgCTTTggCCg 2001 CCgCCCAgTC CTgCTCgCTT CgCTACTTgg AgCCACTATC gACTACgCgA2051 TCATggCgAC CACACCCgTC CTgTggATCT ATCgAATCTA AATgTAAgTT 2101AAAATCTCTA AATAATTAAA TAAgTCCCAg TTTCTCCATA CgAACCTTAA 2151 CAgCATTgCggTgAgCA TCT AgA CCTTCAA CAgCAgCCAg ATCCATCACT                    XbaI2201 gCTTggCCAA TATgTTTCAg TC CCTCAgg A gTTACgTCTT gTgAAgTgAT                         Bsu36I 2251 gAACTTCTgg AAggTTgCAg TgTTAACTCCgCTgTATTgA CgggCATATC 2301 CgTACgTTgg CAAAgTgTgg TTggTACCgg AggAgTAATCTCCACAACTC 2351 TCTggAgAgT AggCACCAAC AAACACAgAT CCAgCgTgTT gTACTTgATC2401 AACATAAgAA gAAgCATTCT CgATTTgCAg gATCAAgTgT TCAggAgCgT 2451ACTgATTggA CATTTCCAAA gCCTgCTCgT AggTTgCAAC CgATAgggTT 2501 gTAgAgTgTgCAATACACTT gCgTACAATT TCAACCCTTg gCAACTgCAC 2551 AgCTTggTTg TgAACAgCATCTTCAATTCT ggCAAgCTCC TTgTCTgTCA 2601 TATCgACAgC CAACAgAATC ACCTgggAATCAATACCATg TTCAgCTTgA 2651 gCAgAAggTC TgAggCAACg AAATCTggAT CAgCgTATTTATCAgCAATA 2701 ACTAgAACTT CAgAAggCCC AgCAggCATg TCAATACTAC ACAgggCTgA2751 TgTgTCATTT TgAACCATCA TCTTggCAgC AgTAACgAAC TggTTTCCTg 2801gACCAAATAT TTTgTCACAC TTAggAACAg TTTCTgTTCC gTAAgCCATA 2851 gCAgCTACTgCCTgggCgCC TCCTgCTAgC ACgATACACT TAgCACCAAC 2901 CTTgTgggCA ACgTAgATgACTTCTggggT AAgggTACCA TCCTTCTTAg 2951 gTggAgATgC AAAAACAATT TCTTTgCAACCAgCAACTTT ggCAggAACA 3001 CCCAgCATCA gggAAgTggA AggCAgAATT gCggTTCCACCAggAATATA 3051 gAggCCAACT TTCTCAATAg gTCTTgCAAA ACgAgAgCAg ACTACACCAg3101 ggCAAgTCTC AACTTgCAAC gTCTCCgTTA gTTgAgCTTC ATggAATTTC 3151CTgACgTTAT CTATAgAgAg ATCAATggCT CTCTTAACgT TATCTggCAA 3201 TTgCATAAgTTCCTCTgggA AAggAgCTTC TAACACAggT gTCTTCAAAg 3251 CgACTCCATC AAACTTggCAgTTAgTTCTA AAAgggCTTT gTCACCATTT 3301 TgACgAACAT TgTCgACAAT TggTTTgACTAATTCCATAA TCTgTTCCgT 3351 TTTCTggATA ggACgACgAA gggCATCTTC AATTTCTTgTgAgg AggCCT                                                  StuI 3401TAgAAACgTC AATTTTgCAC AATTCAATAC gACCTTCAgA AgggACTTCT 3451 TTAggTTTggATTCTTCTTT AggTTgTTCC TTggTgTATC CTggCTTggC 3501 ATCTCCTTTC CTTCTAgTgACCTTTAgggA CTTCATATCC AggTTTCTCT 3551 CCACCTCgTC CAACgTCACA CCgTACTTggCACATCTAAC TAATgCAAAA 3601 TAAAATAAgT CAgCACATTC CCAggCTATA TCTTCCTTggATTTAgCTTC 3651 TgCAAgTTCA TCAgCTTCCT CCCTAATTTT AgCgTTCAAC AAAACTTCgT3701 CgTCAAATAA CCgTTTggTA TAAgAACCTT CTggAgCATT gCTCTTACgA 3751TCCCACAAgg TgCTT CCATg g CTCTAAgAC CCTTTgATTg gCCAAAACAg                 NcoI 3801 gAAgTgCgTT CCAAgTgACA gAAACCAACA CCTgTTTgTTCAACCACAAA 3851 TTTCAAgCAg TCTCCATCAC AATCCAATTC gATACCCAgC AACTTTTgAg3901 TTCgTCCAgA TgTAgCACCT TTATACCACA AACCgTgACg ACgAgATTgg 3951TAgACTCCAg TTTgTgTCCT TATAgCC TCC ggA ATAgACT TTTTggACgA                              BspEI 4001 gTACACCAgg CCCAACgAgTAATTAgAAgA gTCAgCCACC AAAgTAgTgA 4051 ATAgACCATC ggggCggTCA gTAgTCAAAgACgCCAACAA AATTTCACTg 4101 ACAgggAACT TTTTgACATC TTCAgAAAgT TCgTATTCAgTAgTCAATTg 4151 CCgAgCATCA ATAATggggA TTATACCAgA AgCAACAgTg gAAgTCACAT4201 CTACCAACTT TgCggTCTCA gAAAAAgCAT AAACAgTTCT ACTACCgCCA 4251TTAgTgAAAC TTTTCAAATC gCCCAgTggA gAAgAAAAAg gCACAgCgAT 4301 ACTAgCATTAgCgggCAAgg ATgCAACTTT ATCAACCAgg gTCCTATAgA 4351 TAACCCTAgC gCCTgggATCATCCTTTggA CAACTCTTTC TgCCAAATCT 4401 AggTCCAAAA TCACTTCATT gATACCATTATACggATgAC TCAACTTgCA 4451 CATTAACTTg AAgCTCAgTC gATTgAgTgA ACTTgATCAggTTgTgCAgC 4501 TggTCAgCAg CATAgggAAA CACggCTTTT CCTACCAAAC TCAAggAATT4551 ATCAAACTCT gCAACACTTg CgTATgCAgg TAgCAAgggA AATgTCATAC 4601TTgAAgTCgg ACAgTgAgTg TAgTCTTgAg AAATTCTgAA gCCgTATTTT 4651 TATTATCAgTgAgTCAgTCA TCAggAgATC CTCTACgCCg gACgCATCgT 4701 ggCCggCATC ACCggCgCCACAggTgCggT TgCTggCgCC TATATCgCCg 4751 ACATCACCgA TggggAAgAT CgggCTCgCCACTTCgggCT CATgAgCgCT 4801 TgTTTCggCg TgggTATggT ggCAggCCCC gTggCCgggggACTgTTggg 4851 CgCCATCTCC TTgCATgCAC CATTCCTTgC ggCggCggTg CTCAACggCC4901 TCAACCTACT ACTgggCTgC TTCCTAATgC AggAgTCgCA TAAgggAgAg 4951CgTCgAgTAT CTATgATTgg AAgTATgggA ATggTgATAC CCgCATTCTT 5001 CAgTgTCTTgAggTCTCCTA TCAgATTATg CCCAACTAAA gCAACCggAg 5051 gAggAgATTT CATggTAAATTTCTCTgACT TTTggTCATC AgTAgACTCg 5101 AACTgTgAgA CTATCTCggT TATgACAgCAgAAATgTCCT TCTTggAgAC 5151 AgTAAATgAA gTCCCACCAA TAAAgAAATC CTTgTTATCAggAACAAACT 5201 TCTTgTTTCg CgAACTTTTT CggTgCCTTg AACTATAAAA TgTAgAgTgg5251 ATATgTCggg TAggAATggA gCgggCAAAT gCTTACCTTC TggACCTTCA 5301AgAggTATgT AgggTTTgTA gATACTgATg CCAACTTCAg TgACAACgTT 5351 gCTATTTCgTTCAAACCATT CCgAATCCAg AgAAATCAAA gTTgTTTgTC 5401 TACTATTgAT CCAAgCCAgTgCggTCTTgA AACTgACAAT AgTgTgCTCg 5451 TgTTTTgAgg TCATCTTTgT ATgAATAAATCTAgTCTTTg ATCTAAATAA 5501 TCTTgACgAg CCAAggCgAT AAATACCCAA ATCTAAAACTCTTTTAAAAC 5551 gTTAAAAggA CAAgTATgTC TgCCTgTATT AAACCCCAAA TCAgCTCgTA5601 gTCTgATCCT CATCAACTTg AggggCACTA TCTTgTTTTA gAgAAATTTg 5651CggAgATgCg ATATCgAgAA AAAggTACgC TgATTTTAAA CgTgAAATTT 5701 ATCTCAAgATCgCggCCgCg ATCTCgAATA ATAACTgTTA TTTTTCAgTg 5751 TTCCCgATCT gCgTCTATTTCACAATACCA ACATgAgTCA gCTTATCgAT 5801 gATAAgCTgT CAAACATgAg AATTAATTCgATgATAAgCT gTCAAACATg 5851 AgAAATCTTg AAgACgAAAg ggCCTCgTgA TACgCCTATTTTTATAggTT 5901 AATgTCATgA TAATAATggT TTCTTAgACg TACgTCAggT ggCACTTTTC5951 ggggAAATgT gCgCggAAcc CCTATTTgTT TATTTTTCTA AATACATTCA 6001AATATgTATC CgCTCATgAg ACAATAACCC TgATAAATgC TTCAATAATA 6051 TTgAAAAAggAAgAgTATgA gTATTCAACA TTTCCgTgTC gCCCTTATTC 6101 CCTTTTTTgC ggCATTTTgCCTTCCTgTTT TTgCTCACCC AgAAACgCTg 6151 gTgAAAgTAA AAgATgCTgA AgATCAgTTgggTgCACgAg TgggTTACAT 6201 CgAACTggAT CTCAACAgCg gTAAgATCCT TgAgAgTTTTCgCCCCgAAg 6251 AACgTTTTCC AATgATgAgC ACTTTTAAAg TTCTgCTATg TggCgCggTA6301 TTATCCCgTg TTgACgCCgg gCAAgAgCAA CTCggTCgCC gCATACACTA 6351TTCTCAgAAT gACTTggTTg AgTACTCACC AgTCACAgAA AAgCATCTTA 6401 CggATggCATgACAgTAAgA gAATTATgCA gTgCTgCCAT AACCATgAgT 6451 gATAACACTg CggCCAACTTACTTCTgACA ACgATCggAg gACCgAAggA 6501 gCTAACCgCT TTTTTgCACA ACATgggggATCATgTAACT CgCCTTgATC 6551 gTTgggAAcc ggAgCTgAAT gAAgCCATAC CAAACgACgAgCgTgACACC 6601 ACgATgCCTg CAgCAATggC AACAACgTTg CgCAAACTAT TAACTggCgA6651 ACTACTTACT CTAgCTTCCC ggCAACAATT AATAgACTgg ATggAggCgg 6701ATAAAgTTgC AggACCACTT CTgCgCTCgg CCCTTCCggC TggCTggTTT 6751 ATTgCTgATAAATCTggAgC CggTgAgCgT gggTCTCgCg gTATCATTgC 6801 AgCACTgggg CCAgATggTAAgCCCTCCCg TATCgTAgTT ATCTACACgA 6851 CggggAgTCA ggCAACTATg gATgAACgAAATAgACAgAT CgCTgAgATA 6901 ggTgCCTCAC TgATTAAgCA TTggTAACTg TCAgACCAAgTTTACTCATA 6951 TATACTTTAg ATTgATTTAA ATTgTAAACg TTAATATTTT gTTAAAATTC7001 gCgTTAAATT TTTgTTAAAT CAgCTCATTT TTTAACCAAT AggCCgAAAT 7051CggCAAAATC CCTTATAAAT CAAAAgAATA gACCgAgATA gggTTgAgTg 7101 TTgTTCCAgTTTggAACAAg AgTCCACTAT TAAAgAACgT ggACTCCAAC 7151 gTCAAAgggC gAAAAACCgTCTATCAgggC gATggCCCAC TACgTgAACC 7201 ATCACCCTAA TCAAgTTTTT TggggTCgAggTgCCgTAAA gCACTAAATC 7251 ggAACCCTAA AgggAgCCCC CgATTTAgAg CTTgACggggAAAgCCggCg 7301 AACgTggCgA gAAAggAAgg gAAgAAAgCg AAAggAgCgg gCgCTAgggC7351 gCTggCAAgT gTAgCggTCA CgCTgCgCgT AACCACCACA CCCgCCgCgC 7401TTAATgCgCC gCTACAgggC gCgTAAAAgg ATCTAggTgA AgATCCTTTT 7451 TgATAATCTCATgACCAAAA TCCCTTAACg TgAgTTTTCg TTCCACTgAg 7501 CgTCAgACCC CgTAgAAAAgATCAAAggAT CTTCTTgAgA TCCTTTTTTT 7551 CTgCgCgTAA TCTgCTgCTT gCAAACAAAAAAACCACCgC TACCAgCggT 7601 ggTTTgTTTg CCggATCAAg AgCTACCAAC TCTTTTTCCgAAggTAACTg 7651 gCTTCAgCAg AgCgCAgATA CCAAATACTg TCCTTCTAgT gTAgCCgTAg7701 TTAggCCACC ACTTCAAgAA CTCTgTAgCA CCgCCTACAT ACCTCgCTCT 7751gCTAATCCTg TTACCAgTgg CTgCTgCCAg TggCgATAAg TCgTgTCTTA 7801 CCgggTTggACTCAAgACgA TAgTTACCgg ATAAggCgCA gCggTCgggC 7851 TgAACggggg gTTCgTgCACACAgCCCAgC TTggAgCgAA CgACCTACAC 7901 CgAACTgAgA TACCTACAgC gTgAgCATTgAgAAAgCgCC ACgCTTCCCg 7951 AAgggAgAAA ggCggACAgg TATCCggTAA gCggCAgggTCggAACAggA 8001 gAgCgCACgA gggAgCTTCC AgggggAAAC gCCTggTATC TTTATAgTCC8051 TgTCgggTTT CgCCACCTCT gACTTgAgCg TCgATTTTTg TgATgCTCgT 8101CAggggggCg gAgCCTATgg AAAAACgCCA gCAACgCggC CTTTTTACgg 8151 TTCCTggCCTTTTgCTggCC TTTTgCTCAC ATgTTCTTTC CTgCgTTATC 8201 CCCTgATTCT gTggATAACCgTATTACCgC CTTTgAgTgA gCTgATACCg 8251 CTCgCCgCAg CCgAACgACC gAgCgCAgCgAgTCAgTgAg CgAggAAgCg 8301 gAAgAgCgCC TgATgCggTA TTTTCTCCTT ACgCATCTgTgCggTATTTC 8351 ACACCgCATA TggTgCACTC TCAgTACAAT CTgCTCTgAT gCCgCATAgT8401 TAAgCCAgTA TACACTCCgC TATCgCTACg TgACTgggTC ATggCTgCgC 8451CCCgACACCC gCCAACACCC gCTgACgCgC CCTgACgggC TTgTCTgCTC 8501 CCggCATCCgCTTACAgACA AgCTgTgACC gTCTCCgggA gCTgCATgTg 8551 TCAgAggTTT TCACCgTCATCACCgAAACg CgCgAggCAg

[0254] TABLE 254 restriction map of pD2pick(MFαPrePro::EPI-HNE-3)Non-cutters AflII ApaI AscI AvaI AvrII BamHI BglII BssHII BstEII MluIPacI PmlI RsrII SacII SfiI SnaBI SpeI XhoI XmaI Cutters, 3 or fewersites AatII 1 1098 EcoRV 2 1885 5660 AflIII 1 8179 Esp3I(BsaI) 2 31208530 AgeI 1 1436 EspI(Bpu1102I) 1 597 AlwNI 3 2828 2852 7765 FspI 2 19606629 ApaLI 3 6182 7865 8363 HindIII 3  885 1717 1729 AseI 3  591 58226678 HpaI 2 1017 2272 BglI 3  284 2717 6730 KpnI 2 2323 2934 BsaAI 27191 8427 MacI 2 2204 3789 BsgI 2 2545 4494 NcoI 1 3766 BsiWI 3 15682301 5929 NdeI 1 8357 BspDI 2 1723 5795 NgoMI 2 4702 7294 BspEI 1 3978NheI 2 1929 2875 BspMI 1 4576 NotI 3    6 5712 8596 Bst1107I 1 8408 NruI1 5208 BstBI(AsuII) 1  945 NsiI 2  684 1241 BstXI 3  711 2765 2896 PflMI2  196 1302 Bsu36I 1 2223 PmeI 1  420 DraIII 2 3754 7188 PpuMI 2  1424339 EagI 3    7 5713 8597 PstI 1 6608 Eam1105I 2 5077 6849 PvuI 1 6482Ecl136I 1  216 PvuII 2 1600 4497 Eco47III 2 1932 4795 SacI 1  216 EcoNI3 3433 4923 5295 SalI 1 3312 EcoRI 1 1383 ScaI 2 1360 6371 SphI 1 4863SspI 3 2806 6047 6983 StuI 1 3395 Tth111I 1 8432 XbaI 1 2168 XcmI 1 711

[0255] TABLE 400 Amino-acid Sequence of ITI light chain (SEQ ID NO. 077)                     111111 111122            12345 6789012345 678901           avlpq eeegsgggql vtevtk22222222333333333344444444445555555555666666666677777772345678901234567890123456789012345678901234567890123456KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTC    |——————|————————————|——————|—————————|——|             |————————————|——————|           |                             |————————————————|    77788    78901   rtvaa                   111111111111111111111111111111111111888888889999999999000000000011111111112222222222333333234567890123456789012345678901234567890123456789012345CNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVP|——————|————————————|——————|——————————|——|         |————————————|——————|            |                         |—————————————————|                                        111111111111                                        333344444444                                        678901234567                                        gdgdeellrfsn

[0256] TABLE 602 Physical properties of hNE inhibitors derived fromKunitz domains k_(on) k_(off) Par- # Mol Predicted K_(D) (10⁶/ (10⁻⁶/Protein ent Residues Wt pI (pM) (M/s) s) EPI-HNE-1 BPTI 58 6359 9.10 2.03.7 7.4 EPI-HNE-2 BPTI 62 6759 4.89 4.9 4.0 20. EPI-HNE-3 ITI- 56 617910.04 6.2 8.0 50. D2 EPI-HNE-4 ITI- 56 6237 9.73 4.6 10.6 49. D2

[0257] TABLE 603 SUMMARY OF PURIFICATION OF EPI-HNE-2 VolumeConcentration Total Activity STAGE (ml) (mg/ml) (mg) (mg/A₂₈₀) HARVEST3,300 0.70 2.31 <0.01 30K ULTRA- 5,000 0.27 1.40 <0.01 FILTRATIONFILTRATE 5K ULTRA- 1,000 1.20 1.20 0.63 FILTRATION RETENTATE AMMONIUM300 2.42 0.73 1.05 SULFATE PRECIPITATE IEX pH 6.2 98 6.88 0.67 1.03ELUATE EPI-HNE-3, 50 13.5 0.68 1.04 LOT 1

[0258] TABLE 604 SUMMARY OF PURIFICATION OF EPI-HNE-3 CONCENTRA- VOLUMETION TOTAL ACTIVITY STAGE (ml) (mg/ml) (mg) (mg/A₂₈₀) HARVEST 3,1000.085 263 nd 30K ULTRA- 3,260 0.055 179 0.007 FILTRATION FILTRATE FIRSTIEX: 180 0.52 94 0.59 pH 6.2 ELUATE AMMONIUM 100 0.75 75 0.59 SULFATEPRECIPITATE IEX pH 9 60 1.01 60 0.59 ELUATE EPI-HNE-3, 26 1.54 40 0.45LOT 1

[0259] TABLE 605 K_(I) VALUES OF EPI-HNE PROTEINS FOR VARIOUS HUMANSERUM SERINE PROTEASES Inhibitor: EPI- EPI- EPI- EPI- Enzyme HNE-1 HNE-2HNE-3 HNE-4 Human Neutrophil  2 pM   5 pM   6 pM  5 pM Elastase HumanSerum Plasmin  >6 μM >100 μM >100 μM >90 μM Human Serum Kallikrein >10μM >100 μM >100 μM >90 μM Human Serum Thrombin >90 μM >100 μM >100μM >90 μM Human Urine Urokinase >90 μM >100 μM >100 μM >90 μM HumanPlasma Factor X_(a) >90 μM >100 μM >100 μM >90 μM Human Pancreatic ˜10μM  ˜10 μM  ˜30 μM ˜10 μM Chymotrypsin

[0260] TABLE 607 PEY-33 which produces EPI-HNE-2 Elapse Fermenter TimeCell Density Activity in supernatent Hours:minutes (A₆₀₀) (mg/l) 41:0989 28 43:08 89 57 51:54 95 92 57:05 120 140 62:43 140 245 74:45 160 36087:56 170 473 98:13 190 656 102:25  200 678 109:58  230 710

[0261] TABLE 608 PEY-43 Which produces EPI-HNE-3 Elapse Fermenter TimeCell Density Activity in supernatent Hours:minutes (A₆₀₀) (mg/l) 44:30107 0.63 50:24 70 9.4 52:00 117 14. 62:00 131 28. 76:00 147 39. 86:34200 56. 100:27  185 70. 113:06  207 85.

[0262] TABLE 610 Inhibitory properties of EPI-HNE-2 μl of EPI-HNE-2solution Percent residual hNE added activity 0. 101.1 0. 100.0 0. 100.00. 100.0 0. 100.0 0. 98.9 10. 82.9 20. 71.8 30. 59.5 40. 46.2 50. 39.255. 32.2 60. 22.5 65. 23.5 70. 15.0 75. 10.4 80. 8.6 85. 4.8 90. 1.4 95.2.0 100. 2.5 120. 0.2 150. 0.2 200. 0.04

[0263] TABLE 611 hNE inhibitory properties of EPI-HNE-3 μl of EPI-HNE-2Percent residual solution added hNE activity 0. 101.2 0. 100.0 0. 100.00. 100.0 0. 100.0 0. 98.8 10. 81.6 20. 66.9 30. 53.4 40. 38.0 50. 27.655. 21.5 60. 13.0 65. 11.0 70. 7.9 75. 3.8 80. 3.3 85. 2.1 90. 1.8 100.1.6 110. 0.8 120. 0.7 160. 0.6 200. 0.2

[0264] TABLE 612 pH stability of Kunitz-domain hNE inhibitors PercentResidual hNE Inhibitory Activity Incubation pH EPI-HNE-1 EPI-HNE-2EPI-HNE-3 EPI-HNE-4 1.0 102 98 97 98 2.0 100 97 97 100 2.6 101 3.0 100101 100 96 4.0 98 101 102 94 5.0 100 5.5 99 99 109 6.0 100 103 99 6.5 99100 7.0 93 103 103 93 7.5 87 109 8.0 96 84 83 8.5 104 68 86 9.4 100 4440 10.0 98 102 27 34

[0265] TABLE 620 Stability of hNE inhibitory proteins to oxidation byChloramine-T Table 620 Molar Ratio Percent Residual hNE-InhibitoryActivity CHL-T: EPI- EPI- EPI- EPI- α1 anti Inhibitor HNE-1 HNE-2 HNE-3HNE-4 trypsin SLPI 0 100 100 100 100 100 100 0.25 94 0.29 93 0.30 97 .48102 .50 102 97 100 85 .59 82 .88 73 .95 100 1.0 102 97 100 41 1.2 65 1.498 1.5 95 1.9 102 2.0 102 2.1 7 2.4 48 3.0 97 100 3.8 94 4.0 95 5.0 94100 5.2 7 5.9 18 9.5 95 10. 98 97 104 10.4 >5 12. 15 19. 92 30. 100 10050. 94 100 #quenched reactions is shown as a percentage of the activityobserved with no added oxidant. Proteins and concentrations in theoxidation reactions are: EPI-HNE-1, (5 μM); EPI-HNE-2, (10 μM);EPI-HNE-3, (10 μM); EPI-HNE-4, (10 μM); API, (10 μM); and SLPI, (8.5μM).

[0266] TABLE 630 Temperature stability of EPI-HNE proteins TemperatureResidual hNE Inhibitory Activity (° C.) EPI-HNE-1 EPI-HNE-2 EPI-HNE-3EPI-HNE-4 0 97 101 96 100 23 100 103 105 103 37 100 97 99 98 45 103 52101 100 55 99 98 65 94 95 87 69 82 75 100 80 101 79 85 106 63 93 88 5795 64 48

[0267] TABLE 711 Mutations that are likely to improve the affinity of aKunitz domain for hNE Most Preferred X18F; [X15I (preferred), X15V];Highly Preferred [X16A (Preferred), X16G]; [X17F (preferred), X17M,X17L, X17I, X17L]; [{X19P, X19S} (equally preferred), X19K, X19Q]; X37G;X12G; Preferred X13P; X20R; X21Y; X21W; [X34V (preferred), X34P]; [X39Q,X39M]; [X32T, X32L]; [X31Q, X31E, X31V]; [X11T, X11A, X11R]; [X10Y,X10S, X10V]; [X40G, X40A]; X36G;

[0268] TABLE 720 M13_III signal::Human_LACI-D2::mature_M13_III DNA hasSEQ ID NO. 078, amino-acid sequence has SEQ ID NO. 079. DNA is linearand in vivo it is double stranded. Amino-acid sequence is of a proteinthat is processed in vivo by cleavage after Ala⁻¹; the entire geneencodes an amino-acid sequence that continues to give a functional M13III protein.  M   K   K   L   L   F  −18 −17 −16 −15 −14 −13|atg|aaG|aaG|ctt|ctc|ttc|    |       |     HindIII A  I   P   L   V   V   P   F   Y   S   G   A −12 −11 −10 −9  −8  −7  −6  −5  −4  −3  −2  −1|gcc|att|cct|ctg|gtg|gta|cct|ttc|tat|tcc|ggc | BstXI        | | KpnI |             |  KasI |  |       XcmI       |     K   P   D   F   C   F   L   E   E   D   P     1   2   3   4   5   6   7   8   9   10  11|gcc|aag|cct|gac|ttc|tgc|ttc|ctc|gag|gag|gat|ccc|                        | XhoI  |       |  XmaI |G    I   C   R   G   Y   I   T   R   Y   F 12 13  14  15  16  17  18  19  20  21  22ggg|att|tgc|cgc|ggt|tat|att|acg|cgt|tat|ttc|           | SacII|         | MluI  | Y   N   N   Q   T   K   Q   C   E   R 23  24  25  26  27  28  29  30  31  32|tat|aat|aac|cag|act|aag|caa|tgt|gag|cgg|                       | BsrDI| | BsrI  | F   K   Y   G   G   C   L   G   N   M 33  34  35  36  37  38  39  40  41  42|ttc|aag|tat|ggt|ggt|tgc|cta|ggt|aat|atg|                       | AvrII|  N   N   F   E   T   L   E   E   C   K 43  44  45  46  47  48  49  50  51  52|aac|aac|ttc|gag|act|cta|gaa|gag|tgt|aag|                    | XbaI | N   I   C   E   D   G   G   A    53  54  55  56  57  58 100 101 |aac|ata|tgt|gag|gat|ggt|ggt|gct    | NdeI |  E   T   V   E   S102 103 104 105 106 |gag|act|gtt|gag|tct|    |  DrdI        |

[0269] TABLE 725 Synthetic laci-d1 with sites for cloning into displayvector DNA has SEQ ID NO. 080, amino-acid sequence has SEQ ID NO.081    A   A   E   M   H   S   F   C   A   F   K   A                1   2   3   4   5   6   7   8   95′-gcg|gcc|gag|atg|cat|tcc|ttc|tgc|gct|ttc|aaa|gct    |EagI  |  | NsiI  |    D    D   G   P   C   K   A   I   M   K   R  10    11  12  13  14  15  16  17  18  19  20|gat|  |gaC|ggT|ccG|tgt|aaa|gct|atc|atg|aaa|cgt|          | RsrII |              | BspHI|    F   F   F   N   I   F   T   R   Q   C   21  22  23  24  25  26  27  28  29  30  |ttc|ttc|ttc|aac|att|ttc|acG|cgt|cag|tgc|                          | MlUI  |    E   E   F   I   Y   G   G   C   E   G   N   Q   31  32  33  34  35  36  37  38  39  40  41  42  |gag|gaA|ttC|att|tac|ggt|ggt|tgt|gaa|ggt|aac|cag|      | EcoRI |                       | BstEII |            N   R   F   E   S   L   E   E           43  44  45  46  47  48  49  50          |aac|cgG|ttc|gaa|tct|ctA|gag|gaa|            |     | BstBI |  | XbaI |             | AgeI |    C   K   K   M   C   T   R   D   G   A   51  52  53  54  55  56  57  58  59  101  |tgt|aag|aag|atg|tgc|act|cgt|gac|ggc|gcc                                  | KasI  |

[0270] TABLE 730 LACI-D1 hNE Library DNA has SEQ ID NO. 082, amino-acidsequence has SEQ ID NO. 083    A   A   E   M   H   S   F   C   A   F   K                1   2   3   4   5   6   7   85′-gcg|gcc|gag|atg|cat|tcc|ttc|tgc|gct|ttc|aaa|     |EagI  |  | NsiI  |                                         S        T|N                             T|N    C|R K|R                             I|M    S|G S|A                             Q|H    Y|H E|G     H|R             F|L     L|P  A  D|N  D   G  P|L  C  V|I A|G I|V  F  K|R  R  9  10  11  12  13  14  15  16  17  18  19  20|gct|NRt|RVS|ggT|cNt|tgt|Rtt|gSt|Ntc|ttc|MNS|cgt|     C    Y|W   F|L   F   F   N   I   F   T   R   Q   C   21  22  23  24  25  26  27  28  29  30  |tDS|ttc|ttc|aac|att|ttc|acG|cgt|cag|tgc|                          | MluI  |        Q       Q                   Q       L|P     L|P                 L|P       T|K     T|K                 T|K   L|Q V|I     V|E                 V|M         E|G   E|V E|A  F  I|A  Y   G   G   C  E|A G|A  N  Q|R   31  32  33  34  35  36  37  38  39  40  41  42  |SWG|VHA|ttC|VHA|tac|ggt|ggt|tgt|VHG|gSt|aac|SRG|            N   R   F   E   S   L   E   E           43  44  45  46  47  48  49  50          |aac|cgG|ttc|gaa|tct|ctA|gag|gaa|            |     | BstBI |  | XbaI |             | AgeI |    C   K   K   M   C   T   R   D   G   A   51  52  53  54  55  56  57  58  59  101  |tgt|aag|aag|atg|tgc|act|cgt|gac|ggc gcc                                  | KasI  |

[0271] TABLE 735 LACI-D2 hNE Library DNA has SEQ ID NO. 084; amino-acidsequence has SEQ ID NO. 085                                             C|R                                             S|G                                             Y|H  G   A   K   P   D   F   C   F   L   E   E  D|N −2  −1   1   2   3   4   5   6   7   8   9   10|ggc|gcc|aag|cct|gac|ttc|tgc|ttc|ctc|gag|gag|NRt||  KasI |                       | XhoI  |  P|H T|N                             I|N K|R     H|R             F|L     Q|M S|A     P|L             I|V     L|H      C E|G     N|S             Y|H     K|P     F|L D|Q  G  I|T  C  V|I G|A N|D  F  T|R  R  Y|W  F  11  12  13  14  15  16  17  18  19  20  21  22|VVS|ggg|MNt|tgc|Rtt|gSt|NWt|ttt|MNS|cgt|tDS|ttc|                                     Q|G                                     L|P                                     T|K                                     V|I                                 L|Q E|A  Y   N   N   Q   A   K   Q   C  E|V  R  23  24  25  26  27  28  29  30  31  32|tat|aat|aac|cag|Gct|aag|caa|tgt|SWg|VNA|                |      | BsrDI|                 | EspI   |     Q|L                 Q|P      P|T                 T|K         R|G     V|E                 V|M         K|E     I|A                 E|A         L|Q  F   K   Y   G   G   C   L  G|A  N  M|V  33  34  35  36  37  38  39  40  41  42|ttc|VHA|tat|ggt|ggt|tgc|VHG|gSt|aat|VBg|  N   N   F   E   T   L   E   E   C   K  43  44  45  46  47  48  49  50  51  52|aac|aac|ttc|gag|act|cta|gaa|gag|tgt|aag|                    | XbaI |  N   I   C   E   D   G   G   A   53  54  55  56  57  58 100 101|aac|ata|tgt|gag|gat|ggt|ggt|gct|    | NdeI |   E   T   V   E   S 102 103 104 105 106 |gag|act|gtt|gag|tct|    |  DrdI        |

[0272] TABLE 790 Amino acids preferred in hNE-inhibiting Kunitz domainsPosition Allowed amino acids 5 C 10 YSV, (NA) 11 TAR, (QP) 12 G 13 P,(VALI) 14 C 15 IV 16 AG 17 FM, ILV (A) 18 F 19 PS, QK 20 R 21 YW, (F) 30C 31 QEV, (AL) 32 TL, (PSA) 33 F 34 VP 35 Y 36 G 37 G 38 C 39 MQ 40 G, A41 N highly preferred 42 G preferred, A allowed 45 F 51 C 55 C

[0273] CITATIONS ALBR83a: Albrecht et al., Hoppe-Seyler's Z Physiol Chem(1983), 364:1697-1702. ALBR83b: Albrecht et al., Hoppe-Seyler's ZPhysiol Chem (1983), 364:1703-1708. ALTM91: Altman et al., ProteinEngineering 4(5)593-600 (1991). AUER87: Auerswald et al., Biol ChemHoppe-Seyler (1987), 368:1413-1425. AUER88: Auerswald et al., Bio ChemHoppe-Seyler (1988), 369(Supplement):27-35. AUER89: Auerswald et al., UKPatent Application GB 2,208,511 A. AUER90: Auerswald et al., U.S. Pat.No. 4,894,436 (16 Jan. 1990). AUSU87: Ausubel et al., Editors. CurrentProtocols in Molecular Biology, Greene Publishing Associates andWiley-Interscience, Publishers: John Wiley & Sons, New York, 1987.BERN93: Berndt et al., J Mol Biol (1993) 234 (3) p 735-50. BRIN90: BiolChem Hoppe-Seyler (1990) 371(Suppl)43-52. Brinkmann and Tschesche.BRIN91: Eur J Biochem (1991) 202(1)95-99. Brinkmann et al. CAMP82: JClin Invest 70:845-852 (1982) Campbell et al. CAMP88: J Cell Biol106:667-676 (1988) Campbell et al. CANT89: Cantor JO, and GM Turino, pp.159-168 in Elastin and Elastase, Vol. II, Editors L Robert and WHornebeck, CRC Press, Boca Raton, Fla., 1989. DIAR90: Diarra-Mehrpour etal., Eur J Biochem (1990), 191:131-139. DIGA89: Digan et al., (1989)Bio/Technology 7:160ff. ENGH89: Enghild et al., J Biol Chem (1989),264:15975-15981. GEBH86: Gebhard, W, and K Hochstrasser, pp. 389-401 inBarret and Salvesen (eds.) Protease Inhibitors (1986) Elsevier SciencePublishers BV (Biomedical Division). GEBH90: Gebhard et al., Biol ChemHoppe-Seyler (1990), 371, suppl 13-22. GOLD86 Am Rev Respir Dis134:49-56 (1986) Goldstein, W, and G Doering. GREG93: Gregg et al.,Bio/Technology (1993) 11:905-910. HEID86 Heidtmann, H, and J Travis, pp.441-446 in Proteinase Inhibitors, Editors Barrett and Salvesen, ElsevierScience Publishers BV, Amsterdam, 1986. HYNE90: Hynes et al.,Biochemistry (1990), 29:10018-10022. KAUM86: Kaumerer et al., NucleicAcids Res (1986), 14:7839-7850. MCEL91 The Lancet 337:392-4 (1991)McElvaney et al. MCWH89 Biochem 28:5708-5714 (1989) McWherter et al.NORR93: Norris et al., WIPO Application 93/14123. ODOM90: Odom, L, Int JBiochem (1990), 22:925-930. ROBE92: Roberts et al., (1992) Proc NatlAcad Sci USA 89(6)2429-33. SALI90 TIBS 15:435-9 (November 1990) Salier,J-P. SAMB89: Sambrook et al., Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory, 1989. SCHA87: Schagger,H. and G. von Jagow (1987) Analytical Biochemistry 166:368ff. SCHE67:Schecter and Berger, Biochem Biophys Res Comm (1967) 27:157-162. SELL87:Selloum et al., Biol Chem Hoppe-Seyler (1987), 368:47-55. SKAR92:Skarzynski, T, J Mol Biol (1992) 224(3)671-83. SPRE94: Sprecher et al.,Proc Natl Acad Sci USA 91:3353-3357 (1994). STOL90: Stoll and Blanchard(1990) Methods in Enzymology 182:24ff. SWAI88: Swaim, MW, and SV Pizzo,Biochem J (1988), 254:171-178. TRAB86: Traboni, C, R Cortese, NucleicAcids Res (1986), 14(15)6340. TRAV88 Am J Med 84(6A)37-42 (1988) Travis.VEDV91: Vedvick et al., J Ind Microbiol (1991) 7:197-201. WAGN92: Wagneret al., Biochem Biophys Res Comm (1992) 186:1138-1145.

1. A non-naturally occurring protein which inhibits human neutrophilelastase and which is a protein comprising at least the core sequence ofa non-naturally occurring Kunitz domain, said Kunitz domain being moresimilar in sequence to the core sequence 26-76 of ITI-D1 than to thecore sequence 5-55 of BPTI, when its cysteines are aligned with those ofBPTI and ITI-D1, but said domain differing from ITI-D1 in that at leastone of the following conditions applies: (a) the residue correspondingto BPTI residue 15 and ITI-D1 residue M36 is Val or Ile, (b) the residuecorresponding to BPTI residue 16 and ITI-D1 residue G37 is Ala, (c) theresidue corresponding to BPTI residue 18 and ITI-D1 residue T39 is Phe,(d) the residue corresponding to BPTI residue 19 and ITI-D1 residue S40is Pro, (e) the residue corresponding to BPTI residue 1 and ITI-D1residue K22, if any, is Arg, (f) the residue corresponding to BPTIresidue 2 and ITI-D1 residue E23, if any, is Pro, or (g) the residuecorresponding to BPTI residue 4 and ITI-D1 residue S25, if any, is Phe.2. The protein of claim 1 which differs from human ITI-D1 at least oneof the positions corresponding to BPTI positions 15-20.
 3. The proteinof claim 1 where, in said Kunitz domain, BPTI positions 1-4 areArg-Pro-Asp-Phe (residues 1-4 of SEQ ID NO:17).
 4. The protein of claim1 where the said Kunitz domain the residue corresponding to BPTIposition 31 is Glu.
 5. The protein of claim 1 where the said Kunitzdomain the residue corresponding to BPTI position 31 is Gin.
 6. Theprotein of claim 1 where the said Kunitz domain the residuecorresponding to BPTI position 34 is Val.
 7. The protein of claim 1where in said Kunitz domain the residue corresponding to BPTI position 4is Phe.
 8. The protein of claim 1 where in said Kunitz domain theresidue corresponding to BPTI position 2 is Pro.
 9. The protein of claim1 where the said Kunitz domain the residue corresponding to BPTIposition 1 is Arg.
 10. The protein of claim 1 where the said Kunitzdomain the residue corresponding to BPTI position 26 is Ala.
 11. Theprotein of claim 1 where the said Kunitz domain the residuecorresponding to BPTI position 18 is Phe.
 12. The protein of claim 1where in said Kunitz domain the residue corresponding to BPTI position15 is Val or Ile, 16 is Ala or Gly, 17 is Met or Phe and 19 is Pro orSer.
 13. The protein of claim 1 which has an affinity for HNE such thatits KD is less than 10⁻⁸ M.
 14. The protein of claim 1 which has anaffinity for HNE such that its KD is less than 10⁻⁹ M.
 15. The proteinof claim 1 which has an affinity for HNE such that its KD is less than10⁻¹¹ M.
 16. The protein of claim 1 wherein both conditions (a) and (c)apply.
 17. The protein of claim 16 wherein condition (d) also applies.18. The protein of claim 1 wherein conditions (e)-(g) apply.
 19. Theprotein of claim 16 wherein conditions (e)-(g) also apply.
 20. Theprotein of claim 17 wherein conditions (e)-(g) also apply.
 21. Theprotein of claim 1 where said Kunitz domain is a reference domainselected from the group consisting of BITI-E7-1222, AMINO1 (SEQ IDNO:22), AMINO2 (SEQ ID NO:23), MUTP1 (SEQ ID NO:24), BITI-E7-141 (SEQ IDNO:17), MUTT26A (SEQ ID NO:18), MUTQE (SEQ ID NO:19), and MUT1619 (SEQID NO:20) or a Kunitz domain comprising an amino acid sequence whichotherwise differs from the core sequence of one or more of saidreference domains solely by one or more class A and/or one or more classB substitutions as set forth in Table
 65. 22. The protein of claim 1where said non-naturally occurring Kunitz domain is a reference domainselected from the group consisting of BITI-E7-1222, AMINO1, AMINO2,MUTP1, BITI-E7-141, MUTT26A, MUTQE, and MUT1619 in Table 220 or a kunitzdomain comprising an amino acid sequence which differs from the coresequence of one or more of said reference domains solely by one or moreclass A substitutions as set forth in Table
 65. 23. The protein of claim1 where the core sequence of said Kunitz domain consists of an aminoacid sequence identical to that of the core sequence of a referencedomain selected from the group consisting of BITI-E7-1222, AMINO1,AMINO2, MUTP1, BITI-E7-141, MUTT26A, MUTQE, and MUT1619 in Table 220.24. The protein of claim 1 where said Kunitz domain is selected from thegroup consisting of BITI-E7-1222, AMINO1, AMINO2, MUTP1, BITI-E7-141,MUTT26A, MUTQE, and MUT1619 in Table
 220. 25. The protein of claim 24where said protein further comprises at least a functional portion of acoat protein of a filamentous phage, sufficient to cause display of saidprotein on the surface of a filamentous phage particle if said proteinis expressed, together with the other proteins of said phage, in a cellcapable of assembling said particles.
 26. The protein of claim 25 wheresaid coat protein is the one corresponding in said filamentous phage tothe gene III protein of M13 phage.
 27. The protein of claim 1 which isidentical to a protein selected from the group consisting ofBITI-E7-1222, AMINO1, AMINO2, MUTP1, BITI-E7-141, MUTT26A, MUTQE, andMUT1619 in Table
 220. 28. The protein of claim 1 where said protein isBITI-E7-141.
 29. The protein of claim 1 where said protein is MUTT26A(SEQ ID NO:18).
 30. The protein of claim 1 where said protein is MUTQE(SEQ ID NO:19).
 31. The protein of claim 1 where said protein is MUT1619(SEQ ID NO:20).
 32. The protein of claim 1 where said Kunitz domain isnot identical in amino acid sequence to any of the Kunitz domain aminoacid sequences set forth in Table
 13. 33. A method of inhibiting humanneutrophil elastase (HNE) which comprises contacting the HNE with aninhibitor effective amount of a protein of any one of claims 1, 12, and14-23.
 34. A method of inhibiting harmful human neutrophil elastaseactivity in a subject which comprises administering to the subject aninhibitorily effective amount of a protein of any one of claims 1, 12and 14-23.
 35. A method of treating emphysema in a subject whichcomprises administering to the subject a therapeutically effectiveamount of a protein of claim
 1. 36. A method of treating cystic fibrosisin a subject which comprises administering to the subject atherapeutically effective amount of a protein of claim 1.